Functional lipid constructs

ABSTRACT

The invention relates to functional lipid constructs and their use in diagnostic and therapeutic applications, including serodiagnosis, where the functional moiety is carbohydrate, peptide, chemically reactive group, conjugator or fluorophore.

TECHNICAL FIELD

The invention relates to methods for effecting qualitative andquantitative changes in the functional moieties expressed at the surfaceof cells and multi-cellular structures, and functional lipid constructsfor use in such methods. In particular, the invention relates tofunctional lipid constructs and their use in diagnostic and therapeuticapplications, including serodiagnosis, where the functional moiety is acarbohydrate, peptide, chemically reactive group, conjugator orfluorophore.

BACKGROUND ART

The ability to effect qualitative and quantitative changes in thefunctional moieties expressed at the surface of liposomes, cells andmulti-cellular structures provides for a range of diagnostic andtherapeutic applications. A functional moiety may be a carbohydrate,peptide, chemically reactive group (e.g. maleimide), conjugator (e.g.biotin) or fluorophore (e.g. fluorescein).

The specification accompanying international application numberPCT/NZ2005/000052 (publication number WO 2005/090368) describes thepreparation of carbohydrate-lipid constructs for use in methods ofeffecting qualitative and quantitative changes in the level ofcarbohydrates expressed at the surface of cells and multi-cellularstructures. The use of the constructs to prepare quality control cellsfor use in blood grouping and diagnostics is described.

The specification accompanying international application numberPCT/NZ2006/000245 (publication number WO 2007/035116) describes anothermethod for the preparation of carbohydrate-lipid constructs where thecarbohydrate is the polymer hyaluronic acid. The use of the constructsto modify embryos to promote association with endometrial cells isdescribed.

The specification accompanying international application numberPCT/NZ2007/000256 (publication number WO 2008/030115) describes thepreparation of fluorophore-lipid constructs. The use of the constructsin methods of fluorescently labeling cells is described.

Known methods of effecting changes in the peptides expressed at thesurface of cells include gene manipulation, chemical modification ofmembrane peptides, and “cell surface painting” using lipid anchors suchas GPI (Legler et al (2004), McHugh et al (1995), Medof et al (1996),Metzner et al (2008), Morandat et al (2002), Premkumar et al (2001),Ronzon et al (2004), Skountzou et al (2007)).

In addition to these methods of effecting changes of endogenouslyexpressed peptides, exogenously prepared peptides may be coupled tolipids of the membrane utilising biotin-avidin conjugation. Biotin bindsto the tetrameric protein avidin with a dissociation constant (K_(D)) ofthe order 10⁻¹⁵ mol/L. This strong binding is exploited in a number oflaboratory applications.

In these laboratory applications biotin is linked to a molecule such asa carbohydrate or a peptide. The preferential binding of avidins tobiotin is exploited in a number of isolation or separation applicationsin addition to the coupling of peptides to the lipids of membranes.

The specification accompanying international application no.PCT/NZ02/00214 (publication no. WO 03/039074) describes a “two-stepmethod” of localizing an antigen such as a peptide to the surface ofcells. In the method the biotinylated glycoside (BioG) is contacted witha suspension of cells for a time and at a temperature sufficient toallow the BioG molecules to incorporate via their diacyl lipid tailsinto the cell membrane of the cells.

An exogenously prepared avidinylated peptide may then be localized tothe surface of the BioG modified cells by contacting the peptide withthe modified cells. Alternatively, an exogenously prepared biotinylatedpeptide may be localized to the surface of the modified cells via abiotin-avidin bridge.

In either alternative of the “two-step method” the amount of peptidelocalized to the surface of the cells may be controlled by controllingthe concentration, time and temperature at which the BioG molecules arecontacted with the suspension of cells to provide the modified cells.However, the utility of the method is limited by the availability anddispersibility of BioG in biocompatible media such a saline.

The specification accompanying international application no.PCT/NZ2005/000052 (publication no. WO 2005/090368) describes a “one-stepmethod” of localizing carbohydrate antigen to the surface of cells. The“one-step method” utilizes carbohydrate-lipid constructs that aredispersible in biocompatible media and can therefore be used to preparemodified cells without loss of vitality. However, a method of preparingpeptide-lipid constructs with comparable dispersibility in biocompatiblemedia and of general applicability to peptides has not been described.

Relatively little work has been performed on the coupling of peptides tophospholipids as individual components prior to their incorporation inself assembling lipid structures, such as liposomes. However, a varietyof standard techniques have been described for the covalent coupling ofpeptides to liposome surfaces.

Martin et al (1990) has reviewed methods of attaching moieties includingpeptides, to the surface of liposomes.

Blume et al (1993) describes the coupling of the water solubleGlu-plasminogen to liposomes by the method described by Kung andRedemann (1986). The chemical ECDI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) is used to activate the liposomes prior toincubation of the activated liposome suspension with Glu-plasminogen.Proteo-PEG-coated liposomes with Glu-plasminogen covalently attached tothe ends of the distearylyphosphatidylethanolamine (DSPE)-PEG-COOH areprovided.

Haselgrübler et al (1995) describes a heterobifunctional crosslinkerused to facilitate the preparation of immunoliposomes. The crosslinkeris synthesised from a diamine derivative of poly(ethylene glycol) (PEG,average molecular weight 800 dalton (18mer)). The crosslinker has2-(pyridylthio)propronyl (PDP) and N-hydroxysuccinimide ester (NHS) asfunctional groups.

Ishida et al (2001) describes the preparation of liposomes bearingpolyethylene glycol-coupled transferrin. Transferrin was conjugated viathe terminal carboxyl residue of DSPE-PEG-COOH. The liposomes wereproposed as having utility in in vivo cytoplasmic targeting ofchemotherapeutic agents or plasmid DNAs to target cells.

Massaguer et al (2001) describes the incorporation of a peptide sequence(GGRGRS) and hydrophobic derivatives to the surface of chemicallyactivated liposomes. The incorporation was carried out through thecarboxyl group of N-glutaryl dipalmitoyl phosphatidyl choline (NGPE).

Massaguer et al (2001) noted that considering potential in vivoapplications, where sterility and simplicity would be some of the mostimportant requirements, processes based on chemical reactions on thesurface of liposomes involving extra steps would be more difficult to bescaled up at the industrial level. A hydrophobic derivative of thepeptide sequence was identified as providing optimal properties forincorporation to the surface of liposomes.

Chung et al (2004) describe the antigenic determinant shielding effectof DOPE-PEG incorporated into the membranes of cells and speculatedconcerning the potential of lipid-PEG(n)(s) to regulate biological cellresponses and the extension of this concept to the introduction offunctional molecules at the end of the PEG chain.

Kato et al (2004) describe a method for anchoring of macromolecularproteins into the membranes of living mammalian cells. Adioleylphosphatidylethanolamine (DOPE) derivative coupled withhydrophilic poly(ethylene glycol) (PEG80) was used as the syntheticmembrane anchor. Peptides were conjugated at the distal terminal of thePEG moiety via an amino-reactive N-hydroxysuccinimide derivative of thesynthetic membrane anchor.

The PEG80 moiety facilitated solubilisation of the synthetic membraneanchor in water. As noted by Kato et al (2004) if the anchor isinsoluble in water, undesirable and complicated processes such asliposome preparation and the fusion of liposomes with the cell membranemay be required to anchor the conjugates into the cell membrane.

An additional advantage noted by Kato et al (2004) was that syntheticmembrane anchors with high hydrophile-lipophile balance values(attributable to PEG spacer with a high number of oxyethylene units)were concluded to have no cytolytic activity. However, difficultiesarise in the use of synthetic membrane anchors including a PEG spacerwith a high number of oxyethylene units.

Firstly, the expression of the conjugative peptide or other endogenouscell surface peptides may be masked by the PEG spacer. Secondly, a PEGspacer with a high number of oxyethylene units may elicit non-specificadherence of protein (including antibodies in certain individuals)and/or the non-specific activation of the complement cascade.

Winger et al (1996) describes the conjugation of bromoacetylated DSPEwith a thiol terminated decapeptide comprising at its C-terminus theminimal human thrombin-receptor peptide agonist(HS---SerPheLeuLeuArgAsn).

Hashimoto et al (1986) describes the conjugation of iodoacetylated DSPEwith thiolated compounds.

A need exists for a general method of preparing peptide-lipid constructsthat may be incorporated as individual components in self assemblinglipid structures, such as liposomes, by a “one-step method”. The methodshould desirably provide peptide-lipid constructs that are readilydispersible in biocompatible media and spontaneously incorporate in tothe membranes of cells and multi-cellular structures.

Peptide-lipid constructs with these characteristics are anticipated tohave utility in a range of therapeutic and diagnostic applications,especially serodiagnosis, in addition to the preparation offunctionalized liposomes.

It is an object of this invention to provide functional-lipid constructsthat are dispersible in biocompatible media and spontaneouslyincorporate into the membranes of cells and multi-cellular structures.

It is an object of this invention to provide functional-lipid constructsfor use in the preparation of peptide-lipid constructs that aredispersible in biocompatible media and spontaneously incorporate intothe membranes of cells and multi-cellular structures.

It is an object of this invention to provide peptide-lipid constructsthat are dispersible in biocompatible media and spontaneouslyincorporate into the membranes of cells and multi-cellular structures.

These objects are to be read disjunctively with the object to at leastprovide the public with a useful choice.

DISCLOSURE OF INVENTION

In a first aspect the invention provides a functional lipid construct ofthe structure F-S-L where F is a functional moiety, L is a diacyl or adialkyl lipid, and S is a spacer covalently linking F to L including thesubstructure:

where g is the integer 1, 2 or 3, M is a monovalent cation orsubstituent, and * is other than H.

Preferably, the substructure is:

where h is the integer 1, 2, 3 or 4.

Preferably, g is the integer 2 and h is the integer 1, 2 or 4.

Preferably, M is H or CH₃.

Preferably, L is a diacylglycerophospholipid. More preferably, L isphosphatidylethanolamine.

Preferably, the structure of the functional lipid construct includes thepartial structure:

where v is the integer 3, 4 or 5, M′ is a monovalent cation, and R₁ andR₂ are independently selected from the group consisting of: alkyl oralkenyl substituents of the fatty acids trans-3-hexadecenoic acid,cis-5-hexadecenoic acid, cis-7-hexadecenoic acid, cis-9-hexadecenoicacid, cis-6-octadecenoic acid, cis-9-octadecenoic acid,trans-9-octadecenoic acid, trans-11-octadecenoic acid,cis-11-octadecenoic acid, cis-11-eicosenoic acid or cis-13-docsenoicacid.

Preferably, F is a functional moiety selected from the group consistingof: carbohydrate (glycan), peptide, chemically reactive group,conjugator or fluorophore.

In a first alternative of the first aspect the invention provides afunctional lipid construct of the structure:

where F is a carbohydrate, x is the integer 2, 3 or 4, y is the integer1, 2 or 3, and R₃ is O of a substituted hydroxyl of the carbohydrate.

Preferably, the carbohydrate is a glycan selected from the groupconsisting of: GalNAcα1-3(Fucα1-2)Galβ-; Galα1-3(Fucα1-2)Galβ-;Xylα1-3Glcβ-; Xylα1-3Xylα1-3Glcβ-; Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ-;Galβ1-4(Fucα1-3)GlcNAcβ-; Fucα1-2Galβ1-4(Fucα1-3)GlcNAcβ-;Neu5Acα2-3Galβ1-3GalNAcα-; NeubAcα2-3Galβ1-3(6-HSO₃)GalNAcα-;Neu5Acα2-3Galβ1-3GlcNAcβ-; Neu5Acα2-3Galβ1-3(6-HSO₃)GlcNAcβ-;Neu5Acα2-3Galβ-; GalNAcα1-3Galβ3-4GlcNAc-; Galα1-3Galβ1 4GlcNAc-;Galα1-4Galβ1-4Glc-; Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ-;GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ-; Galα1-3(Fucα1-2)Galα1-4GlcNAcβ-;Fucα1-2Galβ1-4GlcNAcβ-; Galα1-3Galβ1-4GlcNAcβ-; Galα1-4Galβ1-4GlcNAcβ-;Neu5Acα2-3Galβ1-4GlcNAcβ-; Neu5Acα2-3Galα1-4(6-HSO₃)GlcNAcβ-;Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ;Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-;Neu5Ac-α-(2-6′)-Galβ1-4GlcNAcβ-; and Neu5Gc-α-(2-6′)-Galβ1-4GlcNAcβ-.

In a second alternative of the first aspect the invention provides afunctional lipid construct of the structure:

where F is a peptide, w is the integer 1 or 2, and R is S of asubstituted sulfhydryl of a Cys residue of the peptide.

In a third alternative of the first aspect the invention provides afunctional lipid construct of the structure:

where F is the chemically reactive group maleimide and w is the integer1 or 2.

In a fourth alternative of the first aspect the invention provides afunctional lipid construct of the structure:

where F is the conjugator biotin and k is the integer 2, 3 or 4.

In a fifth alternative of the first aspect the invention provides afunctional lipid construct of the structure:

where F is the fluorophore of fluorescein (or one of its derivatives), zis the integer 3, 4 or 5, and R₃ is C of the thiocyanate substituent ofthe isothiocyanate derivative of fluorescein (or one of itsderivatives).

Preferably, the substructure is:

designated MCMG(1);

designated MCMG(2);

designated CMG(1); or

designated CMG(2).

In a second aspect the invention provides a water soluble peptide-lipidconstruct of the structure F-S-L where S is a spacer linked to F via asulphide bond and includes the substructure:

where g is the integer 1, 2 or 3, M is a monovalent cation orsubstituent, and * is other than H.

Preferably, the substructure is:

where h is the integer 1, 2, 3 or 4.

Preferably, g and h are the integer 2.

Preferably, M is H or CH₃.

Preferably, L is a diacylglycerophospholipid. More preferably, L isphosphatidylethanolamine.

Preferably, the structure of the peptide-lipid construct includes thepartial structure:

where v is the integer 3, 4 or 5, M′ is a monovalent cation, and R₁ andR₂ are independently selected from the group consisting of: alkyl oralkenyl substituents of the fatty acids trans-3-hexadecenoic acid,cis-5-hexadecenoic acid, cis-7-hexadecenoic acid, cis-9-hexadecenoicacid, cis-6-octadecenoic acid, cis-9-octadecenoic acid,trans-9-octadecenoic acid, trans-11-octadecenoic acid,cis-11-octadecenoic acid, cis-11-eicosenoic acid or cis-13-docsenoicacid.

Preferably, the structure of the peptide-lipid construct includes thepartial structure:

where w is the integer 1 or 2, and R₃ is S of a substituted sulfhydrylof a Cys residue of the peptide.

In an embodiment of the second aspect the invention provides apeptide-lipid construct of the structure:

where the sum of x and y is greater than 5. Optionally, F is a peptideincluding a proximal terminal sequence (PTS) selected to promotesolubility of the peptide.

In a preferment of this option, the PTS of the peptide is selected fromthe group consisting of:

SerLysLysLysLysGly AlaAlaAlaAla GlySerGlySerGly

Preferably, the Cys residue is a terminal Cys residue of the peptide(Cys).

Preferably, the terminal sequence of the peptide is selected from thegroup consisting of:

GlyLysLysLysLysSerCys AlaAlaAlaAlaCys GlySerGlySerGlyCysCysSerLysLysLysLysGly CysAlaAlaAlaAla CysGlySerGlySerGly

Preferably, the Cys residue is a terminal Cys residue of the peptide atthe carboxy-terminus of the peptide.

Preferably, F is a peptide comprising an epitope of antigens selectedfrom the group consisting of: Glycophorin A, Glycophorin B, or mutationsthereof (including the MNS blood group system). More preferably, F is apeptide selected from the List of Peptides. Most preferably, F is apeptide selected from the group consisting of:

GlnThrAsnAspLysHisLysArgAspThrTyrAlaAlaAlaAlaAla CysGlnThrAsnAspLysHisLysArgAspThrTyrGlySerGlySerGly CysGlnThrAsnAspMetHisLysArgAspThrTyrGlySerGlySerGly CysSerSerGlnThrAsnAspLysHisLysArgAspThrTyrCysThrTyrProAlaHisThrAlaAsnGluValCys ThrTyrProAlaHisThrAlaAsnGluCysProAlaHisThrAlaAsnGluValCys SerGlnThrAsnAspLysHisLysArgAspCysCysThrTyrProAlaHisThrAlaAsnGlu

Preferably, L is a glycerophospholipid selected from the groupconsisting of: 1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine(DOPE) and 1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE).

In an exemplifying first embodiment of the second aspect the inventionprovides a peptide-lipid construct of the structure:

designated DOPE-Ad-CMG(2)-βAla-Ma-PTS-Milt(K) (IX).

In an exemplifying second embodiment of the second aspect the inventionprovides a peptide-lipid construct of the structure:

designated DOPE-Ad-CMG(2)-βAla-Mal-Milt(K,M) (X).

In an exemplifying third embodiment of the second aspect the inventionprovides a peptide-lipid construct of the structure:

where R₁ and R₂ are both (CH₂)₇CHCH(CH₂)₇ and designatedDOPE-Ad-CMG(2)-βAla-Mal-Mur(D14C) (XI).

In an exemplifying fourth embodiment of the second aspect the inventionprovides a peptide-lipid construct of the structure:

where R₁ and R₂ are both (CH₂)₇CHCH(CH₂)₇ and designatedDOPE-Ad-CMG(2)-βAla-Mal-Syph (V8C) (XII).

In a third aspect the invention provides a method of detecting reactiveantibody in the serum of a subject including the steps of:

-   -   Contacting a sample of the serum with a suspension of cells        modified to incorporate a functional lipid construct (F-S-L) of        the first or second alternatives of the first aspect of the        invention or a peptide-lipid construct of the second aspect of        the invention to provide a mixture;    -   Incubating the mixture for a time and at a temperature        sufficient to allow agglutination; and    -   Determining the degree of agglutination of the cells in the        mixture;        where:    -   F is a carbohydrate or peptide comprising an epitope for the        reactive antibody.

Optionally, the method includes the intermediate step of:

-   -   Adding an anti-subject globulin antibody to the mixture prior to        determining the degree of agglutination of the cells of the        mixture.

Preferably, the anti-subject globulin antibody is anti-human globulin(AHG) antibody.

Optionally, where F is a peptide, the method includes the preliminarystep of:

-   -   Adding an amount of the peptide to the sample of the serum;        where the amount of the peptide is sufficient to neutralize        non-specific agglutination or confirm specificity of the        reactive antibody.

Preferably, the reactive antibody is reactive to an antigen selectedfrom the group consisting of: Glycophorin A, Glycophorin B, or mutationsthereof (including the MNS blood group system).

Preferably, the subject is a human.

Preferably, the cells are red blood cells.

In a third aspect the invention provides a method of preparing apeptide-lipid construct of the second aspect of the invention includingthe step of:

-   -   Reacting a peptide including a Cys residue with a functional        lipid construct of the third alternative of the first aspect of        the invention.

In a fourth aspect the invention provides a method of effectingqualitative and quantitative changes in the functional moietiesexpressed at the surface of a cell or a multi-cellular structureincluding the step of:

-   -   contacting the cell or multi-cellular structure with a solution        of a functional lipid construct of the first aspect of the        invention for a time and at a temperature sufficient to allow        the construct to incorporate into the cell or multi-cellular        structure.

In a fifth aspect the invention provides a method of immobilizing one ormore cells or multi-cellular structures including the steps of:

-   -   Contacting the cells or multi-cellular structures with a        solution of constructs of the fourth alternative of the first        aspect of the invention for a time and at a temperature        sufficient to allow an effective amount of the constructs to        incorporate into the cells or multi-cellular structures to        provide modified cells or multi-cellular structures; and    -   Contacting the modified cells or multi-cellular structures with        an avidin-coated substrate capable of being reversibly localized        to a surface.

Preferably, the avidin-coated substrate is selected from the groupconsisting of: avidin-coated magnetic beads.

Preferably, the being reversibly localized to a surface is byapplication of a magnetic field.

In an embodiment of the fifth aspect the invention provides a method ofimmobilizing one or more cells or multi-cellular structures includingthe steps of:

-   -   Contacting the cells or multi-cellular structures with a        dispersion of constructs of the structure:

-   -   for a time and at a temperature sufficient to allow an effective        amount of the construct to incorporate into the cells or        multi-cellular structures to provide modified cells or        multi-cellular structures;    -   Contacting the modified cells or multi-cellular structures with        avidin-coated magnetic beads; and    -   Applying a magnetic field to localize the beads to a surface;        where R₁ and R₂ are both (CH₂)₇CHCH(CH₂)₇.

In a sixth aspect the invention provides a method of promoting theaggregation of a first and second population of cells including thesteps of:

-   -   Contacting the first population of cells with a solution of        constructs of the fourth alternative of the first aspect of the        invention for a time and at a temperature sufficient to allow an        effective amount of the constructs to incorporate into the cells        to provide modified cells of the first population;    -   Contacting the second population of cells with a solution of        constructs of the fourth alternative of the first aspect of the        invention for a time and at a temperature sufficient to allow an        effective amount of the constructs to incorporate into the cells        to provide modified cells of the first population;    -   Contacting the modified cells of one of the populations with an        excess of avidin; and then    -   Contacting the modified cells of the first and second        populations. In an embodiment of the sixth aspect the invention        provides a method of promoting the aggregation of a first and        second population of cells including the steps of:    -   Contacting the first population of cells with a solution of        constructs of the structure

-   -   for a time and at a temperature sufficient to allow an effective        amount of the constructs to incorporate into the cells to        provide modified cells of the first population;    -   Contacting the second population of cells with a solution of the        constructs for a time and at a temperature sufficient to allow        an effective amount of the constructs to incorporate into the        cells to provide modified cells of the first population;    -   Contacting the modified cells of one of the populations with an        excess of avidin; and then    -   Contacting the modified cells of the first and second        populations.

In all aspects of the invention M is typically H⁺, but may be replacedby another cation such as Na⁺, K⁺ or NH₄ ⁺ or monovalent substituentsuch as CH₃. The notation M′ excludes M being a monovalent substituentsuch as CH₃.

For the most part amino acid residues of peptides are identifiedaccording to Table 3 of Appendix 2 of Annex C of the AdministrativeInstructions under the Patent Cooperation Treaty dated 7 Feb. 2007 andin accordance with the convention:

-   -   H₂N-XaaXaaXaa . . . XaaXaaXaa-COOH

In Tables the corresponding one-letter codes for amino acid residues maybe employed to provide Tables of acceptable dimensions.

In the description and claims of the specification the followingacronyms, terms and phrases have the meaning provided:

“Avidins” means the biotin-binding tetrameric protein produced in theoviducts of birds, reptiles and amphibians and deposited in the whitesof their eggs, its biotin-binding homomers and biotin-binding modifiedforms thereof including EXTRAVIDIN™, NEUTRAVIDIN™ and NEUTRALITE™.

“Biotin-binding” means non-covalent binding to the biotin moiety with adissociation constant (Kr) under biocompatible conditions of the order10⁻¹⁵ M.

“Diagnostic marker” means a molecule, the presence of which in a bodyfluid of a subject is diagnostic of a phenotype or pathologicalcondition of the subject.

“Dispersible in biocompatible media” means capable of forming a stable,single phase system in a medium at a concentration sufficient to effectqualitative and quantitative changes in the functional moietiesexpressed at the surface of a cell or a multi-cellular structure withoutloss of vitality.

“(or one of its derivatives)” means a chemical modification of thechemical structure to provide a fluorophore with substantiallyequivalent physico-chemical properties, but modified spectralcharacteristics.

“MNS blood group system” means blood group antigens or epitopes of thoseantigens and mutations which are present on either glycophorin A,glycophorin B or mutations which result in glycophorin A/B hybrids.

“pcv” means packed cell volume.

“Proximal terminal sequence” means that portion of the peptide sequenceproximal to the amino- or carboxy-terminus of the peptide (F).

“Reactive antibody” means an immunoglobulin, the presence of which in abody fluid of a subject is diagnostic of a phenotype or pathologicalcondition of the subject.

“RBC” means red blood cells.

“Water soluble” means a stable, single phase system is formed when theconstruct is contacted with water or saline (such as PBS) at aconcentration of at least 100 μg/ml and in the absence of organicsolvents or detergents. The terms “soluble” and “dispersible” are usedsynonymously.

Exemplifying embodiments of the invention will now be described indetail with reference to the Figures of the accompanying drawings pages.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. ¹H-NMR spectrum of the construct DOPE-Ad-CMG(I)amine (11) (5mg/ml in D₂O/CD₃OD 2:1 δ ppm).

FIG. 2. MALDI-TOF MS spectrum of DOPE-Ad-CMG(I)-βAla-Mal-Syph(V8C) (XII)(FLEX-PC (Bruker), DHB).

FIG. 3. ¹H-NMR spectrum of the constructDOPE-Ad-CMG(I)-βAla-Mal-Syph(V8C) (XII) (7 mg/ml in D₂O/CD₃OD 4:1, pH c.7.5; 600 MHz, 30° C., δ ppm).

FIG. 4. ¹H NMR spectrum of Biotin-CMG(2)-Ad-DOPE (I) (2.5 mg/ml inCD₃OD/D₂O 1:1, δ ppm, 600 MHz).

FIG. 5. ESI-MS spectrum of Biotin-CMG(2)-Ad-DOPE (I) (ThermoFinniganLCQDecaXP (negative mode, 30% MeOH)).

FIG. 6. Fluorescence microscopy of avidin AF labelled red blood cellsmodified with the construct designated Biotin-CMG(2)-Ad-DOPE (I) (1mg/mL) and stored for 14 days.

FIG. 7. Fluorescence microscopy of avidin AF labelled zona-free D3.5pcmurine embryos modified with the construct designatedBiotin-CMG(2)-Ad-DOPE (I) (0.1 mg/mL) (400× magnification).

FIG. 8. Fluorescence confocal microscopy of an avidin AF labelledBiotin-CMG(2)-Ad-DOPE (I) modified murine embryo.

FIG. 9. Attachment of streptavidin beads to spermatozoa followingmodification of the spermatozoa by incubation with the constructdesignated Biotin-CMG(2)-Ad-DOPE (I).

FIG. 10. Retention by streptavidin beads of following modification ofthe RBCs by incubation with the construct designatedBiotin-CMG(2)-Ad-DOPE (I).

FIG. 11. Structure of the construct designated Biotin-CMG(2)-Ad-DOPE.

FIG. 12. RL95-2 monolayers modified with 20, 100 and 500 μg/mLbiotin-CMG(2)-Ad-DOPE or media alone in serum-free media (A, C, E and G)and serum-containing media (B, D, F and H).

FIG. 13. Addition of Avidin Alexa Fluor® 488 to RL95-2 monolayersmodified with biotin-CMG(2)-Ad-DOPE. Fluorescence microscopy images ofcells after addition of Avidin Alexa Fluor® 488 incubated at 37° C. showfluorescence starts to internalize within 4 hr (C) and is present in thecell interior in 24 hr (E). No internalization was observed when cellswere incubated at 4° C. (D, F).

DETAILED DESCRIPTION

The invention resides primarily in conjugating functional moieties to adiacyl or dialkyl lipid (L) via a spacer (S) to provide a construct(F-S-L) that is dispersible in biocompatible media, but will alsospontaneously incorporate into the lipid bilayer of a cell membrane ormulti-cellular structure.

The invention resides secondarily in the use of the selected structuralmotif (CMG) in the applications described and the advantages that accruefrom using this structural motif and derivatives thereof.

Despite the advances in cell surface modification described in thespecifications accompanying the international PCT applications referredto under the heading Background Art, the availability of constructs foruse in the “one-step method”, in particular peptide-lipid constructs,and the availability of BioG for use in the “two-step method”, places alimitation on the broad application of these methods.

For example, a two-step method of localizing peptide antigen to thesurface of cells or multi-cellular structures that avoids the use ofBioG, or other conjugates obtained from biological sources, isdesirable.

Although it was recognized that the biotinylation of thecarbohydrate-lipid constructs described in the specificationaccompanying international application no. PCT/NZ2005/000052 provided asubstitute for BioG in the “two-step method”, it remains desirable to beable to use a biotin-lipid construct that has the favourable propertiesof these biotinylated carbohydrate-lipid constructs and could be used inthe “one-step method”.

In contrast with the preparation of constructs where the function (F) isa carbohydrate, the preparation of constructs where F is a peptidepresents additional technical problems.

Firstly, it is desirable for the peptide (F) ligated to the L-S or S-Lmoiety to be dispersible in water such as a buffered solution ofsolutes, e.g. PBS, or at least a biocompatible solvent.

Overcoming this difficulty may require the selection of a proximalterminal sequence (PTS) to promote solubility without modifying thedesired biological properties of the construct.

Secondly, it is desirable for the peptide-lipid construct to bedispersible in water, or at least a biocompatible buffered solution orserum, according to the requirements of the proposed application (i.e.it is desirable for the construct to be “water soluble” as definedherein).

Overcoming this difficulty requires the selection of a spacer (S) topromote solubility of the construct.

Thirdly, where the proposed application is the modification of cellssuch as red blood cells (RBCs) for use in diagnostic applications, or asquality controls in blood group typing, it is required for the constructto be dispersible in a biocompatible buffered solution withoutparticipating in antigen-antibody cross reactivity not specific to thediagnostic peptide or blood group type antigen.

Satisfying this requirement requires the identification of suitablestructural motifs for the spacer (S) and/or proximal terminal sequence(PTS) when the latter is present.

Where the application is for use in the modification of the surface ofcells or multi-cellular structures (e.g. an embryo) with a view topromoting the association of the modified cell or modifiedmulti-cellular structure with a target surface (e.g. the endometrium)exposing the cell or multi-cellular structure to solvents or bufferedsolutions that are not biocompatible must be avoided.

Fourthly, the presentation of the peptide of the peptide lipid constructat the surface of the modified cell or multi-cellular structure willhave an influence on the extent of cross reactivity with diagnosticmarkers.

The ability to localise peptides to the surface of cells ormulti-cellular structures via a residue proximal to either the N- orC-terminus of the peptide may allow the naturally occurringconfiguration of the peptide sequence relative to the cell surface to beapproximated.

The presentation of the peptide sequence in the tertiary (or quaternary)structure of the parent polypeptide (or protein) may therefore bemimicked. It is contemplated that peptides may be localised to thesurface of cells via multiple residues. For example, where both aresidue proximal to the amino terminus and a residue proximal to thecarboxyl terminus are used to localise the peptide a “looped”configuration of the peptide may be promoted at the surface.

The poly-ethylene glycol (PEG) spacer of known peptide-lipid constructsis selected to provide solubility. However, polymers of PEG mayinterfere with the expression and function of the peptide at thesurface.

The as yet unpublished specification accompanying internationalapplication number PCT/NZ2008/000239 describes the preparation ofpeptide-lipid constructs for use in methods of effecting qualitative andquantitative changes in the level of peptides expressed at the surfaceof cells and multi-cellular structures where an oligomer of ethyleneglycol is used as a spacer covalently linking lipid of the construct tothe peptide moiety. The use of the constructs to prepare cells for usein serodiagnosis is described.

In the peptide-lipid constructs of the present invention the structuralmotif designated CMG is used as a component (S₁) of the spacer (S)covalently linking the lipid (L) and peptide (F). Inclusion of thisstructural motif provides a degree of rigidity to the spacer, distancingthe functional moiety (peptide) of the peptide-lipid construct from thesurface of the modified cell or multi-cellular structure.

It will be recognized that this attribute of the invention may befavourably applied to the development of other functional lipidconstructs as demonstrated here with reference to the use of constructsincluding this structural motif where the functional moiety is acarbohydrate, such as the glycotope of the antigens of the ABO bloodgrouping, a fluorophore such as fluorescein (or one of its derivatives),or a conjugator, such as biotin.

Biotin ([3aS-(3aα, 4β,6aα)]-hexahydro-2-oxo-1H-thieno[3,4-d]imidazole-4-pentanoic acid) is awater soluble vitamin of the B complex, also referred to as vitamin H.Biotin is a growth factor present in minute amounts in every livingcell. The compound plays an indispensible role in numerous naturallyoccurring carboxylation reactions, including the production of fattyacids.

Biotin has a solubility (25° C.) in water of approximately 22 mg/100 mLand approximately 80 mg/100 mL in 95% alcohol. The compound hasincreased solubility in hot water and in dilute alkali, but isrelatively insoluble in other common organic solvents. The ability tolocalize this functional moiety to the surface of cells andmulti-cellular structures provides a number of applications asdemonstrated.

Whilst not wishing to be bound by theory it is believed that theproperties of the functional lipid constructs may be modified andrefined to suit particular applications by selection of the cation (M⁺)or derivation of the free carboxyl groups of the structural motif toprovide modified structural motifs, e.g. by substitution with methyl(CH₃; MCMG).

The properties of the functional-lipid constructs for use in the claimedmethods must be such that they can be readily dispersed in biologicallycompatible media in the absence of solvents or detergents, butincorporate into the lipid bilayer of a membrane when a solution of theconstruct is contacted with a suspension of cells or multi-cellular.

Peptide-lipid constructs with these potentially conflicting propertiesare prepared by selection of other components of the spacer (S) inaddition to the inclusion of the unmodified (CMG) or modified (e.g.MCMG) structural motif and/or the inclusion of a proximal terminalsequence (PTS) in the peptide (F).

The preparation of the peptide-lipid constructs where S is linked to Fvia a sulphide bond formed with a terminal Cys (Cys) residue of thepeptide at the carboxy-terminus of the peptide is preferred as thepeptide is less prone to oxidation.

A range of peptides may therefore be prepared as peptide-lipidconstructs for use in methods of effecting qualitative and quantitativechanges in the levels of peptide expressed at the surface of cells andmulti-cellular structures.

A particular advantage of the biotin-lipid constructs is that theypermit cells or multi-cellular structures to be localized to surfaceswith minimal detriment to the biological activity and viability of thecells or multi-cellular structure.

Examples of the localization of cells to a surface are provided. It willbe noted that where the localization to a surface is achieved by meansof avidin-coated magnetic beads the localization is reversible, therebyproviding the opportunity to control the selection and positioning ofcells on a surface.

The utility of the constructs in sub-cellular fractionation andlocalization of membrane bound organelles to surfaces is contemplated.The utility of the constructs in promoting the aggregation ofpopulations of cells as may be required in the generation of hybridomasis also contemplated.

It will be understood that for a non-specific interaction, such as theinteraction between diacyl- or dialkyl-glycerolipids orglycerophospholipids and a membrane, structural and stereo-isomers ofnaturally occurring lipids can be functionally equivalent.

For example, it is contemplated that diacylglycerol 2-phosphate could besubstituted for phosphatidate (diacylglycerol 3-phosphate). Furthermoreit is contemplated that the absolute configuration of phosphatidate canbe either R or S.

The structural motif (CMG) may be prepared by the method summarized inScheme I and Scheme II to provide the substructures designated MCMG(1)and CMG(2).

Chemistry

The preparation of the structural motif, the preparation offunctional-lipid constructs utilizing this structural motif, and the useof these constructs in chemical and biological applications is describedbelow

Preparation of the Structural Motif Designated CMG Materials and Methods

Acetone, benzene, chloroform, ethylacetate, methanol, toluene ando-xylene were from Chimmed (Russian Federation). Acetonitrile was fromCryochrom (Russian Federation). DMSO, DMF, CF₃COOH, Et₃N,N,N′-dicyclohexylcarbodiimide and N-hydroxysuccinimide were from Merck(Germany). Iminodiacetic acid dimethyl ester hydrochloride was fromReakhim (Russian Federation).

Dowex 50X4-400 and Sephadex LH-20 were from Amersham Biosciences AB(Sweden). Silica gel 60 was from Merck (Germany). Tetraamine(H₂N—CH₂)₄C×2H₂SO₄ was synthesized as described by Litherland et al.(1938). Thin-layer chromatography was performed using silica gel 60 F₂₅₄aluminium sheets (Merck, 1.05554) with detection by charring after 7%H₃PO₉ soaking.

Preparation of{[2-(2-tert-butoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-aceticacid methyl ester (SCHEME I)

To a stirred solution of (methoxycarbonylmethyl-amino)-acetic acidmethyl ester hydrochloride (988 mg, 5 mmol) in DMF (15 ml) were addedBoc-GlyGlyNos (3293 mg, 10 mmol) and (CH₃CH₂)₃N (3475 μL, 25 mmol) wereadded. The mixture was stirred overnight at room temperature and thendiluted with o-xylene (70 ml) and evaporated.

Flash column chromatography on silica gel (packed in toluene, and elutedwith ethyl acetate) resulted in a crude product. The crude product wasdissolved in chloroform and washed sequentially with water, 0.5 M NaHCO₃and saturated KCl.

The chloroform extract was evaporated and the product purified on asilica gel column (packed in chloroform and eluted with 15:1 (v/v)chloroform/methanol). Evaporation of the fractions and drying undervacuum of the residue provided a colourless thick syrup. Yield 1785 mg,(95%). TLC: R_(f)=0.49 (7:1 (v/v) chloroform/methanol).

¹H NMR (500 MHz, [D₆]DMSO, 30° C.) δ, ppm: 7.826 (t, J=5.1 Hz, 1H;NHCO), 6.979 (t, J=5.9 Hz, 1H; NHCOO), 4.348 and 4.095 (s, 2H; NCH₂COO), 3.969 (d, J=5.1 Hz, 2H; COCH ₂NH), 3.689 and 3.621 (s, 3H; OCH₃), 3.559 (d, J=5.9 Hz, 2H; COCH ₂NHCOO), 1.380 (s, 9H; C(CH₃)₃).

Preparation of{[2-(2-tert-butoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-aceticacid

To a stirred solution of{[2-(2-tert-butoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-aceticacid methyl ester (1760 mg, 4.69 mmol) in methanol (25 ml) 0.2 M aqueousNaOH (23.5 ml) was added and the solution kept for 5 min at roomtemperature. The solution was then acidified with acetic acid (0.6 ml)and evaporated to dryness.

Column chromatography of the residue on silica gel (packed in ethylacetate and eluted with 2:3:1 (v/v/v) i-PrOH/ethyl acetate/water)resulted in a recovered{[2-(2-tert-butoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-aceticacid methyl ester (63 mg, 3.4%) and target compound (1320 mg). Theintermediate product was then dissolved in methanol/water/pyridinemixture (20:10:1, 30 ml) and passed through an ion exchange column(Dowex 50X4-400, pyridine form, 5 ml) to remove residual sodium cations.

The column was then washed with the same solvent mixture, the eluantevaporated, the residue dissolved in chloroform/benzene mixture (1:1, 50ml) and then evaporated and dried under vacuum. Yield of 10 was 1250 mg(74%), white solid. TLC: R_(f)=0.47 (4:3:1 (v/v/v) i-PrOH/ethylacetate/water).

¹H NMR (500 MHz, [D₆]DMSO, 30° C.), mixture of cis- and trans-conformersof N-carboxymethylglycine unit c. 3:1. Major conformer; δ, ppm: 7.717(t, J=5 Hz, 1H; NHCO), 7.024 (t, J=5.9 Hz, 1H; NHCOO), 4.051 (s, 2H; NCH₂COOCH₃), 3.928 (d, J=5 Hz, 2H; COCH ₂NH), 3.786 (s, 2H; NCH ₂COOH),3.616 (s, 3H; OCH ₃), 3.563 (d, J=5.9 Hz, 2H; COCH ₂NHCOO), 1.381 (s,9H; C(CH₃)₃) ppm; minor conformer, δ=7.766 (t, J=5 Hz, 1H; NHCO), 7.015(t, J=5.9 Hz, 1H; NHCOO), 4.288 (s, 2H; NCH ₂COOCH₃), 3.928 (d, J=5 Hz,2H; COCH ₂NH), 3.858 (s, 2H; NCH ₂COOH), 3.676 (s, 3H; OCH ₃), 3.563 (d,J=5.9 Hz, 2H; COCH ₂NHCOO), 1.381 (s, 9H; C(CH₃)₃).

Preparation of{[2-(2-tert-Butoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-aceticacid N-oxysuccinimide ester (Boc-Gly₂(MCMGly)Nos)

To an ice-cooled stirred solution of{[2-(2-tert-butoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-aceticacid (1200 mg, 3.32 mmol) and N-hydroxysuccinimide (420 mg, 3.65 mmol)in DMF (10 ml) was added N,N′-dicyclohexylcarbodiimide (754 mg, 3.65mol). The mixture was stirred at 0° C. for 30 min, then for 2 hours atroom temperature.

The precipitate of N,N′-dicyclohexylurea was filtered off, washed withDMF (5 ml), and filtrates evaporated to a minimal volume. The residuewas then agitated with (CH₃CH₂)₂O (50 ml) for 1 hour and an etherextract removed by decantation. The residue was dried under vacuumproviding the active ester (1400 mg, 92%) as a white foam. TLC:R_(f)=0.71 (40:1 (v/v) acetone/acetic acid).

¹H NMR (500 MHz, [D₆]DMSO, 30° C.), mixture of cis- and trans-conformersof N-carboxymethylglycine unit c. 3:2.

Major conformer; δ, ppm: 7.896 (t, J=5.1 Hz, 1H; NHCO), 6.972 (t, J=5.9Hz, 1H; NHCOO), 4.533 (s, 2H; NCH ₂COON), 4.399 (s, 2H; NCH ₂COOCH₃),3.997 (d, J=5.1 Hz, 2H; COCH ₂NH), 3.695 (s, 3H; OCH ₃), 3.566 (d, J=5.9Hz, 2H; COCH ₂NHCOO), 1.380 (s, 9H; C(CH₃)₃).

Minor conformer; δ, ppm: 7.882 (t, J=5.1 Hz, 1H; NHCO), 6.963 (t, J=5.9Hz, 1H; NHCOO), 4.924 (s, 2H; NCH ₂COON), 4.133 (s, 2H; NCH ₂COOCH₃),4.034 (d, J=5.1 Hz, 2H; COCH ₂NH), 3.632 (s, 3H; OCH ₃), 3.572 (d, J=5.9Hz, 2H; COCH ₂NHCOO), 1.380 (s, 9H; C(CH₃)₃).

The active ester (1380 mg) was dissolved in DMSO to provide a volume of6 ml and used as a 0.5 M solution (stored at −18° C.).

Preparation of CMG(2) Diamine

A solution of ethylenediamine (808 mg, 13.47 mmol) and Et₃N (1.87 ml,13.5 mmol) in DMSO (5 ml) was added to a stirred solution ofBoc-Gly₂-(MCM)Gly-OSu (15.42 g, 33.68 mmol) in DMSO (50 ml). Thereaction mixture was stirred for 30 min at ambient temperature andacidified with acetic acid (1.2 ml), then fractionated with SephadexLH-20 column (column volume 1200 ml, eluent—MeOH/water 2:1+0.2% AcOH).Fractions containing compound Boc₂MCMG were combined, solventsevaporated and the residue was concentrated in vacuum. The product wasadditionally purified by silica

gel column chromatography using 2-propanol/ethyl acetate/water (2:6:1)as eluent. Fractions containing pure Boc₂MCMG were combined, solventsevaporated and a residue was dried in vacuum to give target Boc₂MCMG ascolourless foam (8.41 g, 84 t). TLC: R_(f)=0.48 (^(i)PrOH/ethylacetate/water 2:3:1).

¹H NMR (500 MHz, [D₆]DMSO, 30° C.), mixture of conformers ˜3:2: 8.166,8.125, 7.917 and 7.895 (m, total 2H; 2 CONHCH₂), 7.793 (m, 2H;NHCH₂CH₂NH), 7.001 (br. t, 2H; 2 NHCOO), 4.277-3.893 (total 12H; 2CH₂COO, 4 NCH₂CO), 3.690 and 3.635 (s, total 6H; 2 COOCH₃), 3.567 (d,J=5.8 Hz, 4H; 2 CH ₂NHCOO), 3.131 (m, 4H; NHCH ₂CH ₂NH), 1.379 (s, 18H;2 C(CH₃)₃) ppm.

MS, m/z: 769 [M+Na], 785 [M+K].

Trifluoroacetic acid (25 ml) was added to a stirred solution of Boc₂MCMG(4.88 g, 6.535 mmol) in methylene chloride (25 ml) and the solution waskept for 1 h at ambient temperature. Then a reaction mixture wasconcentrated and the residue was evaporated three times with anhydrousMeOH (50 ml), then a residue was extracted three times with Et₂O (100ml) to remove traces of trifluoroacetic acid. The resulted precipitate(as a white solid) was dried to give 5.06 g (˜100%) of MCMG asbis-trifluoroacetic salt. TLC: R_(f)=0.23 (ethanol/water/pyridine/aceticacid 5:1:1:1).

¹H NMR (500 MHz, D₂O, 30° C.), mixture of conformers ˜5:4: 4.400-4.098(total 12H; 2 CH₂COO, 4 NCH₂CO), 3.917 (s, 4H; 2 COCH ₂NH₂), 3.829 and3.781 (s, total 6H; 2 COOCH₃), 3.394 (m, 4H; NHCH ₂CH ₂NH) ppm.

MS, m/z: 547 [M+H], 569 [M+Na], 585 [M+K].

A solution of Boc-Gly₂-(MCM)Gly-OSu (7.79 g, 16.994 mmol) in DMSO (17ml) and Et₃N (2.83 ml, 20.4 mmol) was added to the stirred solution ofMCMG (13) (5.06 g, 6.796 mmol) in DMSO (13 ml). The reaction mixtureafter stirring for 2 h at ambient temperature was acidified with aceticacid (4.0 ml) and fractionated with Sephadex LH-20 column chromatography(column volume 1200 ml, eluent—MeOH/water 2:1+0.2% AcOH). Fractionscontaining pure Boc₂MCMG were combined, solvents evaporated and theresidue was dried in vacuum to give target Boc₂MCMG as colourless foam(8.14 g, 97%). TLC: R_(f)=0.25 (^(i)PrOH/ethyl acetate/water 2:3:1).

¹H NMR (500 MHz, [D₆]DMSO, 30° C.), mixture of conformers: 8.393-7.887(total 6H; 6 CONHCH₂), 7.775 (m, 2H; NHCH₂CH₂NH), 6.996 (br. t, 2H; 2NHCOO), 4.299-3.730 (total 28H; 4 CH₂COO, 10 NCH₂CO), 3.691 and 3.633(s, total 12H; 4 COOCH₃), 3.564 (d, J=5.8 Hz, 4H; 2 CH ₂NHCOO), 3.129(m, 4H; NHCH ₂CH ₂NH), 1.380 (s, 18H; 2 C(CH₃)₃) ppm.

MS, m/z: 1256 [M+Na], 1271 [M+K].

Boc₂MCMG (606 mg, 0.491 mmol) was dissolved in CF₃COOH (2 ml) and thesolution was kept for 30 min at r.t. Trifluoroacetic acid was evaporatedin vacuum and the residue was extracted three times with Et₂O(trituration with 25 ml of Et₂O followed by filtration) to removeresidual CF₃COOH and the obtained white powder was dried in vacuum. Thepowder was dissolved in 4 mL of water and then was freeze-dried. Yieldof MCMG (TFA salt) was estimated as quantitative (actual weight waslarger than theoretical by ˜10% due to stability of hydrates). TLC:R_(f)=0.21 (ethanol/water/pyridine/acetic acid 5:1:1:1).

¹H NMR (500 MHz, [D₂]H₂O, 30° C.), mixture of conformers: 4.430-4.014(total 28H; 4 CH₂COO, 10 NCH₂CO), 3.911 (s, 4H; 2 COCH ₂NH₂), 3.823 and3.772 (s, total 12H; 4 COOCH₃), 3.386 (m, 4H; NHCH ₂CH ₂NH) ppm.

MS, m/z: 1034 [M+H], 1056 [M+Na].

To the solution of MCMG (˜0.49 mmol) in water (20 mL) Et₃N (0.5 mL) wasadded, and the solution was kept for 15 h at r.t. The reaction mixturewas evaporated to dryness and the residue was desalted on Sephadex LH-20column (two methods):

Method A. The residue was dissolved in water (3 ml) and the solution wasdesalted on Sephadex LH-20 column (column volume 250 mL,eluent—MeOH/water 1:1+0.05 M pyridine acetate). Fractions, containingCMG contaminated with salts were combined separately, evaporated and theresidue was desalted again. Combined fractions, containing pure CMG,were evaporated to ˜4 ml volume and freeze dried. Yield of CMG (internalsalt) was 431 mg (90%).

Method B. The residue was dissolved in water (3 ml) and the solution wasdesalted on Sephadex LH-20 column (column volume 250 mL,eluent—MeOH/water 1:1+1% conc. aq. NH₃). Fractions, containing pure CMG,were evaporated to −4 ml volume and freeze dried. The residue (armoniasalt of CMG (16)) was dissolved in ^(i)PrOH/water 1:1 mixture (10 mL),Et₃N (0.2 mL) was added, and the solution was evaporated to dryness.This procedure was repeated twice; the residue was dissolved in 4 mL ofwater and freeze-dried. Yield of the di-Et₃N salt of CMG was 549 mg(95%).

TLC: R_(f)=0.50 (^(i)PrOH/MeOH/acetonitrile/water 4:3:3:4+3% conc. aq.NH₃), or R_(f)=0.43 (^(i)PrOH/EtOH/MeOH/water 1:1:1:1, 0.75M NH₃).

¹H NMR of CMG internal salt (500 MHz, [D₂]H₂O, 30° C.), mixture ofconformers: 4.328-4.006 (total 28H; 4 CH₂COO, 10 NCH₂CO), 3.907 (s, 4H;2 COCH ₂NH₂), 3.381 (m, 4H; NHCH ₂CH ₂NH) ppm.

MS, m/z: 977 [M+H], 999 [M+Na], 1015 [M+K].

Preparation of DOPE-Ad-CMG(I)Amine

DOPE-Ad-CMG(2)amine was prepared from{[2-(2-tert-butoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-aceticacid N-oxysuccinimide ester Boc-Gly₂(MCMGly)Nos according to Scheme III.

Preparation of H₂N-CMG-DOPE

To the intensively stirred solution of CMG (16) (425 mg, 0.435 mmol ofinternal salt) in i-PrOH/water mixture (i-PrOH/water 3:2, 10 mL) the 1 Maq. solution of NaHCO₃ (0.435 mL, 0.435 mmol) and then the solution ofDOPE-Ad-OSu (211 mg, 0.218 mmol) in dichloroethane (0.4 mL) were added.The reaction mixture was stirred for 2 h and then acidified with 0.2 mLof AcOH and evaporated to minimal volume at 35° C. The solid residue wasdried in vacuum (solid foam) and then thoroughly extracted withCHCl₃/MeOH mixture (CHCl₃/MeOH 4:1, several times with 10 mL, TLCcontrol). The extracted residue consisted of unreacted CMG(2) and salts(about 50% of CMG was recovered by desalting of combined the residue anda fractions after chromatography on silica gel according to proceduredescribed in the CMG synthesis.). The combined CHCl₃/MeOH extracts(solution of CMG-Ad-DOPE amine, DOPE-Ad-CMG-Ad-DOPE, N-oxysuccinimideand some CMG) were evaporated in vacuum and dried. The obtained mixturewas separated on silica gel column (2.8×33 cm, ˜200 mL of silica gel inCHCl₃/MeOH 5:1). The mixture was placed on column in MeOH/CHCl₃/watermixture (MeOH/CHCl₃/water 6:3:1+0.5% of pyridine) and the componentswere eluted in a stepwise ternary gradient: MeOH/CHCl₃/water compositionfrom 6:3:1 to 6:2:1 and then to 6:2:2 (all with 0.5% of pyridine).DOPE-Ad-CMG-Ad-DOPE was eluted first (R_(f)=0.75, MeOH/CHCl₃/water3:1:1), followed by desired DOPE-Ad-CMG amine (R_(f)=0.63,MeOH/CHCl₃/water 3:1:1), last eluted was CMG (R_(f)=0.31,MeOH/CHCl₃/water 3:1:1). Fractions, containing pure CMG-Ad-DOPE amine(20) were combined and evaporated to dryness. To remove any lowmolecular weight impurities and solubilised silica gel the residue wasdissolved in ^(i)PrOH/water 1:2 mixture (2 mL), and was passed throughSephadex LH-20 column (column volume 130 mL, eluent—^(i)PrOH/water1:2+0.25% of pyridine). Fractions containing pure CMG-Ad-DOPE amine werecombined and evaporated (˜20% of 2-propanol was added to preventfoaming) to dryness, the residue was dissolved in water (˜4 mL) andfreeze-dried. Yield of CMG-Ad-DOPE amine was 270 mg (68% on DOPE-Ad-OSuor 34% on CMG(16)).

¹H NMR (500 MHz, [D₂]H₂O/[D₄]CH₃OH 2:1, 30° C.): 5.505 (m, 4H; 2CH₂CH═CHCH₂), 5.476 (m, 1H; OCH₂CHCH₂O), 4.626 (dd, J_(gem)=11.6 Hz, 1H;OCHCHCH₂O), 4.461-4.084 (total 37H; 4 CH₂COO, 11 NCH₂CO, OCHCHCH ₂O,OCH₂CH₂N), 4.002 (s, 2H; COCH ₂NH₂), 3.573 (m, 4H; NHCH ₂CH ₂NH),2.536-2.463 (m, total 8H; 4 CH₂CO), 2.197 (m, 8H; 2 CH ₂CH═CHCH ₂),1.807 (m, 8H; 4 CH ₂CH₂CO), 1.480 (m, 40H; 20 CH₂), 1.063 (˜t, J≈6 Hz,6H; 2 CH₃) ppm.

MS, m/z: 1831 [M+H].

Preparation of Functional Lipid Constructs (F-S-L) where F is a Peptide

Preparation of DOPE-Ad-CMG(2)-βAla-Mal-Milt(K,M) (X)

The construct DOPE-Ad-CMG(2)-βAla-Mal-Milt(K,M) (X) was preparedaccording to Scheme IV.

DOPE-Ad-CMG(2)amine was treated with 5-fold excess of3-maleimidopropionic acid oxybenztriazol ester in i-PrOH-water.

Conversion of DOPE-Ad-CMG(2) into was somewhat low maleimido-derivative(about 70%), presumably due to fast hydrolysis of the intermediatepromoted by the amount of organic base, diisopropylethylamine, requiredto be added to keep DOPE-Ad-CMG(2) in solution.

The maleimido-derivative was isolated in 40% yield after gel-permeationchromatography on Sephadex LH-20 (i-PrOH-water, 1:2).

Initially, the conjugation of the maleimido-derivative with peptide wasattempted using i-PrOH-TRIS buffer, pH 8 (1:2), but the intermediateappeared to be almost insoluble in this medium. However, addition ofpyridin (1 μl/mg of intermediate) resulted in immediate dissolution ofreactants and a surprisingly clean and substantially completeconversion.

Notably, although no reducing agent was used to prevent oxidativedeactivation of the peptide, MS analysis of the whole reaction mixturerevealed no traces of S-S dimer.

The desired construct (X) was purified on a Sephadex LH-20 column. Asolubility problem was again encountered as fractions containing (X)were slightly opaque.

This would appear to indicate that the amount of base added to theeluent was insufficient to keep compounds properly charged and solublein the concentration range of 1-5 mg/ml.

The structure of purified construct (X) was unambiguously established byNMR and MS spectra.

NMR spectrum revealed the expected peptide: DOPE ratio as deduced fromthe signal ratio for the most characteristic aromatic and olefinprotons.

According to MS data, almost half of the final product (X) spontaneouslyformed pyroglutamyl derivative ([M−17]⁺ ion).

In MALDI MS spectra of (X) peaks corresponding to unmodified peptide arepresent while the related peaks are absent in ESI-MS spectrum of thesame substance. This is ascribed to facile fragmentation at thethiosuccinimide bond (retro-Michael reaction) under MALDI ionizationconditions (destructive technique).

The general method of preparing peptide-lipid constructs was appliedwith minor modification to the preparation of constructs includingpeptides (F) selected from the following List of Peptides:

List of PeptidesCys(Xaa)_(z)TrpThrProProArgAlaGlnIleThrGlyTyrLeuThrValGlyLeuThrArgArgCys(Xaa)_(z)TrpThrProProArgAlaGlnIleThrGlyTyrArgLeuThrValGlyLeuThrArgArgCys(Xaa)_(z)ValMetTyrAlaSerSerGly Cys(Xaa)_(z)TyrProAlaHisThrAlaAsnGluValMetTyrAlaSerSerGly(Xaa)_(z) CysAspTyrHisArgValMetTyrAlaSerSerGly(Xaa)_(z) CysThrAsnGlyGluThrGlyGlnLeuValHisArgPhe(Xaa)_(z) CysThrAsnGlyGluMetGlyGlnLeuValHisArgPhe(Xaa)_(z) CysAspThrTyrProAlaHisThrAlaAsnGluValSerGlu(Xaa)_(z) CysThrTyrProAlaHisThrAlaAsnGluVal(Xaa)_(z) CysProAlaHisThrAlaAsnGluVal(Xaa)_(z) Cys TyrProAlaHisThrAlaAsnGlu(Xaa)_(z)Cys ThrTyrProAlaHisThrAlaAsn(Xaa)_(z) CysThrTyrProAlaHisThrAlaAsnGlu(Xaa)_(z) CysTyrProAlaHisThrAlaAsnGluVal(Xaa)_(z) CysTyrProAlaHisThrAlaAsnGlu(Xaa)_(z) CysProAlaHisThrAlaAsnGluValSer(Xaa)_(z) CysAspThrTyrProAlaHisThrAlaAsnGlu(Xaa)_(z) CysTyrProAlaHisThrAlaAsnGluValSer(Xaa)_(z) CysSerGlnThrAsnAspLysHisLysArgAsp(Xaa)_(z) CysGlnThrAsnAspLysHisLysArgAspThrTyr(Xaa)_(z) CysGlnThrAsnAspLysHisLysArgAspThrTyrSerSerGlnThrAsnAspMetHisLysArgAspThrTyr(Xaa)_(z)Cys GlnThrAsnAspMetHisLysArgAspThrTyr(Xaa)_(z) CysSerSerGlnThrAsnAspLysHisLysArg(Xaa)_(z) CysSerSerGlnThrAsnAspLysHisLysArgAspThrTyr(Xaa)_(z) CysSerSerGlnThrAsnAspMetHisLysArgAspThrTyr(Xaa)_(z) CysSerSerGlnThrAsnAspLysHisLysArgAspThrTyrSerSerGlnThrAsnAspMetHisLysArgAspThrTyr(Xaa)_(z)Cys GlnThrAsnAspLysHisLysArgAspThr(Xaa)_(z) CysSerGlnThrAsnAspLysHisLysArgAspThr(Xaa)_(z) CysThrAsnAspLysHisLysArgAspThrTyrPro(Xaa)_(z) CysGluGluThrGlyGluThrGlyGlnLeuVal(Xaa)_(z) CysGluGluGluThrGlyGluThrGlyGlnLeu(Xaa)_(z) CysGluThrGlyGluThrGlyGlnLeuValHis(Xaa)_(z) CysSerProProArgArgAlaArgValThr(Xaa)_(z) CysTyrArgTyrArgTyrThrProLysGluLysThrGlyProMetLysGlu(Xaa)_(z) CysTrpGlnProProArgAlaArgIle(Xaa)_(z) CysThrIleThrGlyLeuGluProGlyThrGlu(Xaa)_(z) CysPreparation of Functional Lipid Constructs (F-S-L) where F is a Glycan

Materials and General Methods

Acetone, benzene, chloroform, ethylacetate, methanol, o-xylene, toluene,and 2-propanol were from Khimed (Russian Federation). Acetonitrile wasfrom Cryochrom (Russian Federation). DMSO, DMF, CF₃COOH, Et₃N,N,N′-dicyclohexylcarbodiimide and N-hydroxysuccinimide were from Mercx(Germany). N-methylmorpholin (NMM), 2-maleimidopropionic acid anddisuccimidilcarbonate were from Fluka. Iminodiacetic acid dimethyl esterhydrochloride was from Reakhim (Russian Federation). Molecular sieves(MS 3 Å and 4 Å), trimethylsilyl trifluoromethanesulfonate, andtriphenylphosphine were from Aldrich (Germany). All hydrides,1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), and trichloroacetonitrile werefrom Merck (Germany).

Anhydrous tetrahydrofuran (THF) and diethyl ether (Et₂O) were obtainedby distillation from lithium aluminium hydride (H₄AlLi). Dichloromethanefor glycoside synthesis was dried by distillation from phosphorouspentoxide and calcium hydride and stored over molecular sieves MS 4 Å.Solid reagents were dried for 2 h in vacuo (0.1 mm Hg) at 20 to 40° C.Deacetylation was performed according to Zemplen in anhydrous methanol.The solution of the acetylated compound was treated with 2 M sodiummethylate in methanol up to pH 9. When the reaction was completed, Na⁺ions were removed with cation exchange resin Dowex 50X-400 (H⁺) (Acros,Belgium). The solution was concentrated in vacuo.

Column chromatography was carried out on Silica gel 60 (0.040-0.063 mm,Merck, Germany). Gel chromatography was performed on Sephadex LH-20(Pharmacia, Sweden). Solvents were removed in vacuo at 30 to 40° C.Thin-layer chromatography was performed on Silica gel 60 (Mercx,Germany) precoated plates. Spots were visualized by treating with 5%aqueous orthophosphoric acid and subsequent heating to 150° C. in thecase of carbohydrates or by soaking in ninhydrin solution (3 g/l in 30:1(v/v) butanol-acetic acid) in the case of amines.

Optical rotation was measured on a Jasco DIP-360 digital polarimeter at25° C. Mass spectra were recorded on a Vision-2000 (Thermo Bioanalysis,UK) MALDI-TOF mass spectrometer using dihydroxybenzoic acid as a matrix.¹H NMR spectra were recorded on a Bruker WM spectrometer (500 MHz) at25° C. Chemical shifts (δ, ppm) were recorded relative to D₂O (δ=4.750),CDCl₃ (δ=7.270), and CD₃OD (δ=3.500) as internal standards. The valuesof coupling constants (Hz) are provided. The signals in the ¹H NMRspectra were assigned by suppression of spin-spin interaction (doubleresonance) and 2D-1H,1H-COSY experiments.

Preparation of 3-aminopropyl2-acetamido-2-deoxy-α-D-galactopyranosyl-(1→3)-β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside(GalNAcα1-3Galβ1-4GlcNAc-S₁) (5)

The glycosyl chloride3,4,6-tri-O-acetyl-2-azido-2-desoxy-β-D-galactopyranosylchloride (1) wasprepared according to the method disclosed in the publication of Paulsenet al (1978). The glycosyl acceptor(3-trifluoroacetamidopropyl)-2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-4-O-(2,4-di-O-acetyl-6-O-benzyl-β-D-galactopyranosyl)-β-D-glucopyranoside(2) was prepared according to the method disclosed in the publication ofPazynina et al (2008).

A solution of the glycosyl acceptor (420 mg, 0.5 mmol), silver triflate(257 mg, 1.0 mmol), tetramethylurea (120 μl, 1.0 mmol) and freshlycalcinated molecular sieves 4 Å in dry dichloromethane (20 ml), werestirred at room temperature in darkness for 30 min. Another portion ofsieves 4 Å was added, and a solution of glycosyl chloride (350 mg, 1.0mmol) in dry dichloromethane (3 ml) was added. The mixture was stirredfor 20 h at room temperature. The resin was filtered and washed withmethanol (4×10 ml), then solvent was evaporated. Chromatography onsilica gel (elution with 5-7% isopropanol in chloroform) yielded 407 mg(70%) of the product 3 as a mixture of anomers (α/β=3.0 as determined by¹H-NMR spectroscopy).

A solution of the product 3 (407 mg, 0.352 mmol) in methanol (30 ml) wassubjected to hydrogenolysis over 400 mg 10% Pd/C for 16 h. Then theresin was filtered off, washed with methanol (4×10 ml) and the productconcentrated in vacuum. The dry residue was acetylated with 2:1pyridine-acetic anhydride mixture (6 ml) at 20° C. for 16 h, thereagents being co-evaporated with toluene. Two chromatography steps onsilica gel (elution with 10% isopropanol in ethyl acetate and with 5-10%methanol in chloroform) resulted in 160 mg (42%) of the product 4 and 39mg (10%) of the product 4β.

A solution of 2 M sodium methylate in methanol (200 μl) was added to asolution of the product 4 (160 mg, 0.149 mmol) in dry methanol (4 ml).The solution was evaporated after 1 h, 4 ml water added and the solutionkept for 16 h before being chromatographed on a Dowex-H⁺ column (elutionwith 1 M ammonia). The eluate was evaporated, lyophilized to yield 87.2mg (91%) of the 3-aminopropyltrisaccharide (5).

¹H NMR spectra were recorded on a Bruker BioSpin GmbH spectrometer at303K. Chemical shifts (δ) for characteristic protons are provided in ppmwith the use of HOD (4.750), CHCl₃ (δ 7.270) as reference. Couplingconstants (J) are provide in Hz. The signals in ¹H NMR spectra wereassigned using a technique of spin-spin decoupling (double resonance)and 2D-¹H,¹H-COSY experiments.

The values of optical rotation were measured on a digital polarimeterPerkin Elmer 341 at 25° C.

Mass spectra were registered on a MALDI-TOF Vision-2000 spectrometerusing dihydroxybenzoic acid as a matrix.

4: ¹H-NMR (700 MHz, CDCl₃): 1.759-1.834 (m, 1H, CH sp); 1.853-1.927 (m,1H, CH sp); 1.972, 1.986, 1.996, 2.046, 2.053, 2.087, 2.106, 2.115,2.130, 2.224 (10s, 10×3H, COCH₃); 3.222-3.276 (m, 1H, NCH sp);3.544-3.583 (m, 1H, OCH sp); 3.591-3.661 (m, 2H, NCH sp, H-5a); 3.764(dd≈t, 1H, H-4a, J 8.8); 3.787 (dd, 1H, H-3b, J_(3,4) 3.7, J_(2,3) 9.9);3.836 (br. t, 1H, H-5b, J 7.3); 3.882-3.920 (m, 1H, OCH sp); 3.950 (dd,1H, H-6′c, J_(6′,6″) 10.6, J_(5,6′) 5.2); 4.009 (ddd, 1H, H-2a, J_(1,2)7.9, J_(2,3) 10.0, J_(2,NH) 9.0); 4.076-4.188 (m, 5H, H-6′a, H-6′b,H-6″b, H-5c, H-6″c); 4.415 (d, 1H, H-1a, J_(1,2) 7.9); 4.443 (d, 1H,H-1b, J_(1,2) 7.9); 4.529 (dd, 1H, H-6″a, J_(6′,6″) 12.0, J_(5,6″) 2.5);4.548 (ddd, 1H, H-2c, J_(1,2) 3.4, J_(2,3) 11.6, J_(2,NH) 9.4); 4.893(dd, 1H, H-3c, J_(3,4) 3.1, J_(2,3) 11.6); 5.021 (d, 1H, H-1c, J_(1,2)3.4); 5.039-5.075 (m, 2H, H-3a, H-2b); 5.339 (dd≈d, 1H, H-4b, J 2.9);5.359 (dd, 1H, H-4c, J_(3,4) 2.7, J_(4,5) 0.9); 5.810 (d, 1H, NHAc a,J_(2,NH) 9.0); 6.184 (d, 1H, NRAc c, J_(2,NH) 9.4); 7.310-7.413 (m, 1H,NHCOCF₃ sp). R_(f) 0.31 (EtOAc-iPrOH, 10:1). MS, m/z calculated for[C₄₃H₆₀N₃F₃O₂₅]H⁺: 1076.35, found 1076.

4β: ¹H-NMR (700 MHz, CDCl₃): 1.766-1.832 (m, 1H, CH sp); 1.850-1.908 (m,1H, CH sp); 1.923, 1.969, 1.982, 2.059, 2.071, 2.099 (2), 2.120, 2.136,2.148 (10s, 10×3H, COCH₃); 3.230-3.289 (m, 1H, NCH sp); 3.521 (ddd, 1H,H-2c, J_(1,2) 8.2, J_(2,3) 11.2, J_(2,NH) 7.8); 3.548-3.591 (m, 1H, OCHsp); 3.591-3.648 (m, 2H, NCH sp, H-5a); 3.743 (dd≈t, 1H, H-4a, J 8.6);3.795 (br. t, 1H, H-5b, J 6.5); 3.852 (dd, 1H, H-3b, J_(3,4) 3.6,J_(2,3) 9.9); 3.873-3.923 (m, 2H, H-5c, OCH sp); 4.002 (ddd, 1H, H-2a,J_(1,2) 8.0, J_(2,3) 9.5, J_(2,NH) 8.9); 4.039 (dd, 1H, H-6′b, J_(6′,6″)11.6, J_(5,6′) 6.9); 4.087-4.144 (m, 3H, H-6′a, H-6″b, H-6′c); 4.160(dd, 1H, H-6″c, J_(6′,6″) 11.2, J_(5,6″) 6.0); 4.409, 4.417 (2d≈t, 2×1H,H-1a, H-1b, J 7.6); 4.519 (dd, 1H, H-6″a, J_(6′,6″) 11.8, J_(5,6″) 2.5);4.992 (d, 1H, H-1c, J_(1,2) 8.2); 5.043 (dd, 1H, H-3a, J_(3,4) 8.6,J_(2,3) 9.5); 5.066 (dd, 1H, H-2b, J_(1,2 8.0), J_(2,3) 9.8); 5.350(dd≈d, 1H, H-4c, J 3.2); 5.372 (dd≈d, 1H, H-4b, J 3.4); 5.399 (d, 1H,NHAc c, J_(2,NH) 7.8); 5.449 (dd, 1H, H-3c, J_(3,4) 3.4, J_(2,3)

11.3); 5.856 (d, 1H, NHAc a, J_(2,NH) 8.9); 7.361-7.466 (m, 1H, NHCOCF₃sp). R_(f) 0.24 (EtOAc-iPrOH, 10:1). MS, m/z calculated for[C₄₃H₆₀N₃F₃O₂₅]H⁺: 1076.35, found 1076.

5: ¹H-NMR (700 MHz, D₂O): 1.924-2.002 (m, 2H, CH₂ sp); 2.060, 2.064 (2s,2×3H, NCOCH₃); 3.102 (m≈t, 2H, NCH₂ sp, J 6.8); 3.592-3.644 (m, 1H,H-5a); 3.655 (dd, 1H, H-2b, J_(1,2) 7.9, J_(2,3) 9.9); 3.702 (br. dd,1H, H-5b, J_(5,6′) 3.8, J_(5,6″) 8.2, J_(4,5)≦1); 3.713-3.815 (m, 9H);3.846 (dd, 1H, H-6′a, J_(6′,6″) 12.3, J_(5,6′) 5.3); 3.984-4.062 (m, 4H,OCH sp, H-6″a, H-4b, H-3c); 4.123 (dd≈d, 1H, H-4c, J 2.9); 4.206 (br. t,1H, H-5c, J 6.3); 4.248 (dd, 1H, H-2c, J_(1,2) 3.6, J_(2,3) 11.0); 4.542(2d≈t, 2H, H-1a, H-1b, J 7.4); 5.100 (d, 1H, H-1c, J_(1,2) 3.5). R_(f)0.55 (MeOH-1M aq. Py.AcOH, 5:1). MS, m/z calculated for [C₂₅H₄₅N₃O₁₆]H⁺:644.28; found 644. [α]_(546 nm) +128 (c 0.3; MeCN—H₇O, 1:1).

5β: ¹H-NMR (700 MHz, D₂O): 1.938-1.991 (m, 2H, CH₂ sp); 2.055, 2.062(2s, 2×3H, NCOCH₃); 3.100 (m≈t, 2H, NCH₂ sp, J 6.9); 3.610 (dd, 1H,H-2b, J_(1,2) 7.9, J_(2,3) 9.9); 3.603-3.636 (m, 1H, H-5a); 3.682 (br.dd, 1H, H-5b, J_(5,6′) 4.9, J_(5,6″) 7.8, J_(4,5)≦1); 3.693-3.826 (m,11H); 3.842 (dd, 1H, H-6′a, J_(6′,6″) 12.1, J_(5,6′)5.2); 3.934-3.972(m, 2H, H-4b, H-2c); 4.012 (dd, 1H, H-6″a, J_(6′,6″) 12.2, J_(5,6″)2.0); 4.023-4.057 (m, 1H, OCH sp); 4.175 (dd≈d, 1H, H-4c, J 2.9); 4.478(d, 1H, H-1b, J_(1,2) 7.9); 4.531 (d, 1H, H-1a, J_(1,2) 8.1); 4.638 (d,1H, H-1c, J_(1,2) 8.4). R_(f) 0.48 (MeOH-1M aq. Py.AcOH, 5:1). MS, m/zcalculated for [C₂₅H₄₅N₃O₁₆]H⁺: 644.28; found 644. [α]_(546 nm) +6 (c0.3; MeCN—H₂O, 1:1).

Preparation of 3-aminopropyl-α-D-galactopyranosyl-(1→3)-β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside(Galα1-3Galβ1-4GlcNAc-S₁; Galili) (9)

A mixture of the glycosyl acceptor 2 (500 mg, 0.59 mmol),thiogalactopyranoside 6 (576 mg, 1.18 mmol), NIS (267 mg, 1.18 mmol),anhydrous CH₂Cl₂ (25 ml) and molecular sieves 4 Å (500 mg) was stirredat −45° C. for 30 min under an atmosphere of Ar. A solution of TfOH (21μl, 0.236 mmol) in anhydrous CH₂Cl₂ (0.5 ml) was then added. Thereaction mixture was stirred for 2 h at −45° C. and the temperature wasthen increased to −20° C. over 4 h. The mixture was kept at −20° C.overnight. Then extra amounts of thiogalactopyranoside 6 (144 mg, 0.295mmol), NIS (66 mg, 0.295 mmol) and TfOH (5 μl, 0.06 mmol) were added andthe stirring maintained at −20° C. for 2 h before being allowed toslowly warm up to r.t. (1 h). A saturated aqueous solution of Na₂S₂O₃was then added and the mixture filtered. The filtrate was diluted withCHCl₃ (300 ml), washed with H₂O (2×100 ml), dried by filtration throughcotton wool, and concentrated. Gel filtration on LH-20 (CHCl₃-MeOH)afforded the product3-trifluoroacetamidopropyl-3,4-di-O-acetyl-2,6-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,4-di-O-acetyl-6-O-benzyl-β-D-galactopyranosyl-(1→4)-2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-β-D-glucopyranoside(7) (600 mg, 80%), as a white foam.

¹H NMR (700 MHz, CDCl₃, characteristic signals), δ, ppm: 1.78-1.82 (m,4H, CHCHC, OC(O)CH₃), 1.84-1.90 (m, 1H, CHCHC), 1.91, 1.94, 1.97, 1.98,2.06 (5 s, 5×3H, 4 OC(O)CH₃, NH(O)CH₃), 3.23-3.30 (m, 1H, NCHH),3.59-3.65 (m, 1H, NCHH), 4.05 (m, 1H, H-2^(I)), 4.33 (d, 1H, J_(1,2)7.55, H-1^(I)), 4.40 (d, 1H, J 12.04, PhCHH), 4.42 (d, 1H, J_(1,2) 8.07,H-1^(I)), 4.45 (d, 1H, J 11.92, PhCHH), 4.48 (d, 1H, J 12.00, PhCHH),4.50 (d, 1H, J 12.00, PhCHH), 4.52 (d, 1H, J 12.04, PhCHH), 4.54 (d, 1H,J 12.00, PhCHH), 4.57 (d, 1H, J 12.00, PhCHH), 4.64 (d, 1H, J 11.92,PhCHH), 4.99 (dd≈t, 1H, J 8.24, H-2^(II)), 5.08-5.13 (m, 2H, H-3^(I),H-3^(III)), 5.23 (d, 1H, J_(1,2) 3.31, H-1^(III)), 5.46 (d, 1H, J_(3,4)2.25, H-4^(II)), 5.54 (d, 1H, J_(3,4) 3.11, H-4^(III)), 7.20-7.40 (m,20H, ArH); 7.49-7.54 (m, 1H, NHC(O)CF₃). R_(f) 0.4 (PhCH₃—AcOEt, 1:2).

The product 7 (252 mg, 0.198 mmol) was deacetylated according to Zemplen(8 h, 40° C.), neutralized with AcOH and concentrated. The TLC(CH₃Cl-MeOH, 10:1) analysis of the obtained product showed two spots:the main spot with R_(f) 0.45, and another one on the start line(ninhydrin positive spot) that was an indication of partial loss oftrifluoroacetyl. Therefore, the product was N-trifluoroacetylated bytreatment with CF₃COOMe (0.1 ml) and Et₃N (0.01 ml) in MeOH (10 ml) for1 h, concentrated and subjected to column chromatography on silica gel(CHCl₃-MeOH, 15:1) to afford the product 8 as a white foam (163 mg,77%), R_(f) 0.45 (CH₃Cl-MeOH, 10:1). The product 8 was subjected tohydrogenolysis (200 mg Pd/C, 10 ml MeOH, 2 h), filtered,N-defluoroacetylated (5% Et₃N/H₂O, 3 h) and concentrated.Cation-exchange chromatography on Dowex 50X4-400 (H⁺) (elution with 5%aqueous ammonia) gave the product 9 (90 mg, 981) as a white foam.

¹H NMR (D₂O, characteristic signals), δ, ppm: 1.94-1.98 (m, 2H, CCH₂C),2.07 (s, 3H, NHC(O)CH₃), 3.11 (m, J 6.92, 2H, NCH₂), 4.54 and 4.56 (2d,2H, J_(1,2) 8.06, J_(1,2) 7.87, H-1^(I) and H-1^(II)), 5.16 (d, 1H,J_(1,2) 3.87, H-1^(III)). R_(f) 0.3 (EtOH-BuOH-Py-H₂O—AcOH;100:10:10:10:3).

Preparation of 3-aminopropylα-D-galactopyranosyl-(1→4)-β-D-galactopyranosyl-(1→4)-β-D-glucopyranoside (21) and 2-aminoethyl α-D-galactopyranosyl-(1→4)-β-D-galactopyranosyl-(1→4) -β-D-glucopyranoside (28)(Galα1-4Galβ1-4Glc-S₁; Gb₃-S₁)

The title primary aminoalkyl variants of Gb₃-S₁ were prepared accordingSCHEME VII, SCHEME VIII and SCHEME IX.

Preparation of(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-α-D-glucopyranosyltrichloroacetimidate (10)

Trichloroacetonitrile (12.1 ml, 121 mmol) and DBU (0.45 ml, 3 mmol) wereadded to a solution of 10a (7.68 g, 12.1 mmol) in dry dichloromethane(150 ml) at −5° C. The reaction mixture was stirred at −5° C. for 3.5 hand concentrated in vacuo.

Flash chromatography (2:1 to 1:2 (0.1% Et₃N) toluene-ethyl acetate) ofthe residue provided 10 (6.01 g, 63.9%) as a light yellow foam, R_(f)0.55 (2:1 toluene-acetone).

¹H NMR, CDCl₃: 1.95-2.2 (7s, 21H, 7Ac), 4.49 (d, 1H, J_(1,2)=8.07,H-1b), 4.91 (dd, 1H, J_(3,2)=10.3, J_(3,4)=2.8, H-3b), 5.05 (dd, 1H,J_(2,1)=3.5, J_(2,3)=9.3, H-2a), 5.12 (dd, 1H, J_(2,1)=8.07,J_(2,3)=10.3, H-2b), 5.32 (d, 1H, J_(4,3)=3, J_(4,5)<1, H-4b), 5.52 (t,1H, J_(3,2)=J_(3,4)=9.29, H-3a), 6.48 (d, 1H, J_(1,2)=3.5, H-1a), 8.64(s, 1H, HN═CCCl₃).

Preparation of3-chloropropyl-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside(11)

A mixture of 2.94 g (3.8 mmol) of trichloroacetimidate 10, 0.66 ml (7.5mmol) 3-chloropropanol, 50 ml dichloromethane, and 3 g of molecularsieves MS 4 Å was cooled to −5° C. An 8% solution of BF₃.Et₂O (0.4 mmol)in anhydrous dichloromethane was added drop wise with stirring.

After 30 min, the reaction mixture was filtered, diluted with chloroform(500 ml), and washed with water, saturated sodium hydrocarbonatesolution, and water to pH 7. The washed reaction mixture was dried byfiltration through a cotton layer and concentrated in vacuo.

Column chromatography on Silica gel (elution with 2.5:1 (v/v)toluene-ethyl acetate) resulted in 1.75 g (65%) of lactose derivative(11) as white foam. R_(f) 0.54 (2:1 toluene-acetone), R_(f) 0.50 (4:2:1hexane-chloroform-isopropanol), [α]_(D) −4° (c 1.0, CHCl₃), m/z 712.2(M⁺).

¹H NMR, CDCl₁: 1.95 (br. s, 5H, Ac, —CH₂—), 2.0-2.2 (6s, 18H, 6Ac), 3.52(m, 2H, —CH₂Cl), 3.63 (m, 1H, H-5a), 3.68 (m, 1H, OCHH—), 3.79 (t, 1H,J=9.3, H-4a), 3.88 (m, 1H, H-5b), 3.93-3.98 (m, 1H, OCHH—), 4.05-4.15(m, 3H, H-6a′, H-6b, H-6b′), 4.45 (d, 2H, H-1a, H-1b, J_(2,1)=7.83) 4.47(m, 1H, H-6a), 4.89 (dd, 1H, J_(2,3)=9.3, J_(2,1)=7.82, H-2a), 4.96 (dd,1H, J_(3,2)=10.5, J_(3,4)=3.42, H-3b), 5.11 (dd, 1H, J_(2,3)=10.5,J_(2,1)=7.83, H-2b), 5.21 (t, 1H, J=9.3, H-3a), 5.35 (dd, 1H,J_(4,3)=3.42, J_(4,5)<1).

Preparation of 3-azidopropyl (2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside(12)

A mixture of 2.15 g (3 mmol) of trichloropropylglycoside 11, 0.59 g (9mmol) NaN₃, and 30 ml DMSO was maintained at 80° C. with stirring for 20h. The mixture was then diluted with chloroform (500 ml), washed withwater (4×100 ml), dried by filtration through a cotton layer, andconcentrated in vacuo.

Column chromatography on Silica gel (elution with 8:2:1hexane-chloroform-isopropanol) resulted in 1.96 g (91%) of glycoside(12) as a white foam, R_(f)0.54 (2:1 (v/v) toluene-acetone), R_(f) 0.50(4:2:1 (v/v/v) hexane-chloroform-isopropanol), [α]_(D) −5.4° (c 1.0,CHCl₃), m/z 718.8 (M⁺).

¹H NMR, CDCl₃: 1.85 (m, 2H, —CH ₂—), 1.98-2.2 (7s, 2H, 7Ac), 3.36 (m,2H, —CH ₂N₃), 3.61 (m, 2H, H-5a, OCHH—CH₂—), 3.8 (t, 1H,J_(3,4)=J_(4,5)=9.29, H-4a), 3.85-3.94 (m, 2H, OCHH—CH₂; H-5b),4.05-4.17 (m, 3H, H-6a, H-6a′, H-6b), 4.49 (d, 1H, J_(1,2)=8.07, H-1a),4.5 (m, 1H, H-6b′), 4.51 (d, 1H, J_(1,2)=8.07, H-1b), 4.9 (dd, 1H,J_(2,1)=8.07, J_(2,3)=9.29, H-2a), 4.97 (dd, 1H, J_(3,2)=10.27,J_(3,4)=3, H-3b), 5.12 (dd, 1H, J_(2,1)=8.07, J_(2,3)=10.27, H-2b), 5.2(t, 1H, J_(3,2)=J_(3,4)=9.29, H-3a), 5.36 (dd, 1H, J_(4,3)=3,J_(4,5)<1).

Preparation of 3-azidopropyl(4,6-O-benzylidene-β-D-galactopyranosyl)-(1→4) -β-D-glucopyranoside (13)

The lactoside 12 (1.74 g, 2.4 mmol) was deacetylated according toZemplen and co-evaporated with toluene (2×30 ml). The residue wastreated with α,α-dimethoxytoluene (0.65 ml, 3.6 mmol) andp-toluenesulfonic acid (50 mg, to pH 3) in DMF (20 ml) for 3 h. Thereaction mixture was then quenched with pyridine, concentrated, andco-evaporated with o-xylene.

Column chromatography on Silica gel (elution with 9:1 (v/v)chloroform-isopropanol) and recrystallization (chloroform-methanol)resulted in 0.756 mg (62%) of benzylidene derivative (13). R_(f) 0.6(5:1 chloroform-isopropanol), [α]_(D) −25.7° (c 1.0, methanol), m/z513.4 (M⁺).

¹H NMR, CD₃OD: 2.06 (m, 2H, —CH ₂—), 3.45 (dd, 1H, J_(2,1)=J_(2,3′)=9,H-2a), 3.61 (m, 1H, H-5a), 3.64 (m, 2H, —CH ₂N₃), 3.74-3.9 (m, 6H,OCHH—; H-3a, H-4a; H-2b, H-3b, H-5b), 4.08-4.18 (m, 3H, H-6, H-6a′,OCHH—), 4.34-4.44 (m, 3H, H-6b, H-6b′, H-4b), 4.5 (d, 1H, J_(1,2)=7.9,H-1a), 4.68 (d, 1H, J_(1,2)=8, H-1b), 5.82 (s, 1H, CHPh), 7.55-7.72 (m,5H, CHPh).

Preparation of 3-azidopropyl(4,6-O-benzylidene-3-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside(14)

Sodium hydride in mineral oil (290 mg, 12 mmol) was slowly added in 4 to5 portions to a solution of 13 (726 mg, 1.5 mmol) in DMF (15 m) at 0° C.with stirring. After 1 h, the ice bath was removed and benzyl bromidewas added drop wise. The mixture was stirred overnight. 10 ml ofmethanol was then added. After 1 h, the mixture was diluted withchloroform (500 ml), and washed with water (3×200 ml), dried byfiltration through a cotton layer, concentrated, and co-evaporated invacuo with o-xylene.

Column chromatography on Silica gel (elution with 10:1 toluene-ethylacetate) resulted in 1.24 g (87%) of lactose derivative 14 as whitefoam, R_(f) 0.56 (5:3 (v/v) hexane-ethyl acetate), [α]_(D) +10.8° (c1.0, CHCl₃), m/z 963.8 (M⁺).

¹H NMR, CDCl₂: 1.85 (m, 2H, —CH ₂—), 2.91 (m, 1H, H-5b), 3.33 (m, 1H,H-5a), 3.34-3.42 (m, 4H, H-2a, H-3b, —CH ₂N₃), 3.55-3.62 (m, 2H, OCHH—;H-3a), 3.73 (dd, 1H, J_(2,1)=8, J_(2,3)=10, H-2b), 3.92-3.97 (m, 2H,H-4a, OCHH—), 4.0 (br. d, 1H, J_(4,3)=3.6, H-4b), 4.34 (d, 1H,J_(1,2)=7.9, H-1a), 4.42 (d, 1H, J_(1,2)=8, H-1b), 5.43 (s, 1H, CH(Bd),7.14-7.50 (m, 30H, Ph).

Preparation of 3-azidopropyl(2,3,6-O-tri-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside(15)

Hydrogen chloride in diethyl ether was added to a mixture of 14 (1.24 g,1.3 mmol), sodium cyanoborohydride (0.57 g, 9.1 mmol), and freshlyactivated molecular sieves MS 3 Å (33 g) in anhydrous THF (20 ml) untilthe evolution of gas ceased.

The mixture was stirred for 2 h, diluted with chloroform (300 ml),washed with water, saturated sodium hydrocarbonate solution, and waterto pH 7. The washed mixture was dried by filtration through a cottonlayer and concentrated in vacuo.

Column chromatography on Silica gel (elution with 20:1 to 7:3 (v/v)toluene-ethyl acetate) resulted in 0.91 g (65%) of lactose derivative 15as a white foam, R_(f) 0.42 (9:1 (v/v) toluene-acetone), [α]_(D) +17.8°(c 1.0, CHCl₃), m/z 965.8 (M⁺).

¹H NMR, CDCl₃: 1.85 (m, 2H, —CH ₂—), 2.39 (d, 1H, J=2.2, OH), 4.04 (br.s, 1H, H-4b), 4.34 (d, 1H, J_(1,2)=7.9, H-1a), 4.42 (d, 1H, J_(1,2)=8,H-1b), 7.14-7.50 (m, 30H, Ph).

¹H NMR of acetylated analytical probe 15a, CDCl₃: 1.85 (m, 2H, —CH ₂—),4.34 (d, 1H, J_(1,2)=7.9, H-1a), 4.42 (d, 1H, J_(1,2)=8, H-1b), 5.5 (br.d, 1H, J_(4,3)=3.43, H-4b), 7.14-7.50 (m, 30H, Ph).

Preparation of 3-trifluoroacetamidopropyl(2,3,6-O-tri-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside(16)

A mixture of derivative 15 (0.914 g, 0.94 mmol), triphenylphosphine (0.5g, 1.9 mmol) and THE (10 ml) was stirred for 0.5 h, 100 μl of wateradded, and the mixture stirred overnight. The reaction mixture was thenconcentrated and co-evaporated with methanol. The residue was dissolvedin methanol (15 ml) and triethylamine (30 μl) and methyltrifluoroacetate (0.48 ml, 4.7 mmol) added. The solution was held for 30min and then concentrated.

Column chromatography on Silica gel (elution with 5:1 to 1:1 (v/v)hexane-acetone) resulted in 0.87 g (84%) of lactose derivative 16 aswhite foam, R_(f) 0.49 (9:1 (v/v) hexane-acetone), [α]_(D) +17° (c 1.0,CHCl₃), m/z 1060.1 (M⁺+Na).

¹H NMR, CDCl₃: 1.88 (m, 2H, —CH ₂—), 2.40 (br. s, 1H, OH), 4.05 (br. s,1H, H-4b), 4.36 (d, 1H, J_(1,2)=7.8, H-1a), 4.40 (d, 1H, J_(1,2)=7.6,H-1b), 7.10-7.35 (m, 30H, Ph).

Preparation of 2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyltrichloroacetimidate (18)

A mixture of galactose derivative 17 (2 g, 3.65 mmol),trichloroacetonitrile (1.75 ml, 17.55 mmol), anhydrous potassiumcarbonate (2 g, 14.6 mmol), and dichloromethane (4 ml) was stirred for22 h at room temperature under argon. The mixture was then filteredthrough a Celite layer and concentrated in vacuo. Column chromatographyon Silica gel (elution with 4:1 (v/v) hexane-ethyl acetate (1% Et₃N)resulted in 1.5 g (60%) of 18 as white foam, R_(f) 0.47 (7:3 (v/v)hexane-ethyl acetate containing 1% Et₃N) and 0.46 g (0.8 mmol, 23%) ofthe starting derivative 17, R_(f) 0.27 (7:3 (v/v) hexane-ethyl acetatecontaining 1% Et₃N).

¹H NMR (CDCl₃): 3.60-3.70 (m, 3H, H-3, H-6, H-6′), 3.75 (t, 1H,J_(5,6)=6.30, H-5), 3.98 (d, 1H, J_(4,3)=2.19, H-4), 4.08 (dd, 1H,J_(2,3)=9.73, J_(2,1)=7.95, H-2), 4.42 and 4.47 (ABq, 2H, J=12.00, PhCH₂), 4.63 and 4.95 (ABq, 2H, J=11.51, PhCH ₂), 4.72 (s, 2H, PhCH ₂), 4.80and 4.90 (ABq, 2H, J=10.95, PhCH ₂), 5.74 (d, 1H, J_(1,2)=7.95, H-1),7.22-7.35 (m, 20H, ArH), 8.62 (s, 1H, NH).

Preparation of 3-trifluoroacetamidopropyl(2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl)-(1→4)-(2,3,6-tri-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside(19)

A mixture of lactose derivative 16 (158 mg, 0.153 mmol),trichloroacetimidate 18 (120 mg, 0.175 mmol), molecular sieves MS 4 Å(0.5 g), and dichloromethane (5 ml) was stirred for 30 min at roomtemperature under argon. 0.1 ml of a 1% (v/v) solution of trimethylsilyltrifluoromethanesulfonate in dichloromethane was then added. After 2 h,another 50 mg (0.073 mmol) trichloroacetimidate 18 and 30 μl of a 1%(v/v) solution of trimethylsilyl trifluoromethanesulfonate indichloromethane were added. The reaction mixture was stirred overnightat +4° C., quenched with triethylamine (5 μl), filtered, andconcentrated in vacuo.

Column chromatography on Silica gel (elution with 12:1 to 1:1 (v/v)toluene-ethyl acetate) resulted in 170 mg (72%) of trisaccharide 19;R_(f) 0.56 (4:1 (v/v) toluene-ethyl acetate); [α]_(D) +30.8° (c 1.0,CHCl₃).

¹H NMR, CDCl₁: 1.78-1.89 (m, 2H, —CH ₂—), 4.34 (d, 1H, J_(1,2)=7.8,H-1a), 4.43 (d, 1H, J_(1,2)=7.4, H-1b), 5.06 (d, 1H, J_(1,2)=3.0, H-1c),7.14-7.48 (m, 50H, Ph).

Preparation of 3-trifluoroacetamidopropyl(2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl)-(1→4)-(2,3,6-tri-O-acetyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside(20)

The catalyst 10% Pd/C (10 mg) was added to a solution of the protectedoligosaccharide 19 (73 mg, 0.047 mmol) in methanol (7 ml), the mixturedegassed, and the flask filled with hydrogen. The reaction mixture wasstirred for 1 h, filtered off from the catalyst through a Celite layer,and concentrated in vacuo. The dry residue was dissolved in pyridine (2ml), acetic anhydride (1 ml) added, and the mixture held for 3 h. Thesolvents were then evaporated and residue co-evaporated with toluene(4×2 ml).

Column chromatography on Silica gel (elution with 2:1 hexane-acetone)resulted in 43.5 mg (90%) of trisaccharide 20 as a white foam, R_(f)0.52 (2:1 hexane-acetone), [α]_(D) +30.4° (c 1.0, CHCl₃).

¹H NMR, CDCl₃: 1.87 (2H, m, CH₂); 1.99, 2.05, 2.05, 2.06, 2.07, 2.07,2.09, 2.09, 2.12, and 2.14 (10×3H, 10 s, 10 Ac); 3.37 and 3.52 (2×1H, 2m, 2 CHN); 3.63 (1H, ddd, J_(4,5)=9.8, J_(5,6)=4.9, J_(5,6)=2.0, H-5a);3.72 (1H, m, OCH); 3.77 (1H, ddd≈br. τ, J_(4,5)<1, J_(5,6)=6.8,J_(5,6)=6.1, H-5b); 3.79 (1H, dd, J_(3,4)=9.3, J_(4,5)=9.8, H-4a); 3.87(1H, m, OCH); 4.02 (1H, dd≈br. d, J_(3,4)=2.5, J_(4,5)<1, H-4b); 4.09(1H, dd, J_(5,6)=4.9, J_(6,6)=12.0, H-6a); 4.12 (1H, dd, J_(5,6)=5.6,J_(6,6′)=10.8, H-6c); 4.14 (1H, dd, J_(5,6)=6.8, J_(6,6′)=11.0, H-6b);4.17 (1H, dd, J_(5,6′)=8.6, J_(6,6′)=10.8, H-6′c); 4.45 (1H, dd,J_(5,6′)=6.1, J_(6,6′)=11.0, H-6′b); 4.49 (1H, ddd□br. τ, J_(4,5)<1,J_(5,6)=5.6, J_(5,6′)=8.6, H-5c); 4.50 (1H, d, J_(1,2)=7.8, H-1a); 4.55(1H, d, J_(1,2)=7.8, H-1b); 4.59 (1H, dd, J_(5,6′)=2.0, J_(6,6′)=12.0,H-6′a); 4.76 (1H, dd, J_(2,3)=10.8, J_(3,4)=2.5, H-3b); 4.86 (1H, dd,J_(1,2)=8.1, J_(2,3)=9.5, H-2a); 4.10 (1H, d, J_(1,2)=3.4, H-1c); 5.12(1H, dd, J_(1,2)=7.8, J_(2,3)=10.8, H-2b); 5.19 (1H, dd, J_(1,2)=3.4,J_(2,3)=11.0, H-2c); 5.22 (1H, dd≈τ, J_(2,3)=9.5, J_(3,4)=9.3, H-3a);5.40 (1H, dd, J_(2,3)=11.0, J_(3,4)=3.4, H-3c); 5.59 (1H, dd≈br. d,J_(3,4)=3.4, J_(4,5)<1, H-4c); 7.09 (1H, m, NHCOCF₃).

Preparation of 3-aminopropyl α-D-galactopyranosyl-(1→4)-β-D-galactopyranosyl-(1→4) -β-D-glucopyranoside (Gb₃-sp3) (21)

Sodium methylate (30 μl of 2 M solution in methanol) was added to asolution of trisaccharide (20) (43 mg, 0.042 mmol) in anhydrous methanol(3 ml) and held for 2 h. The solution was then concentrated in vacuo,water (3 ml) added, and the mixture held for 3 h. The mixture was thenapplied to a column (10×50 mm) with Dowex 50X4-400 (H⁺) cation exchangeresin.

The target compound was eluted with 1 M aqueous ammonia and the eluantconcentrated in vacuo. Lyophilization from water provided trisaccharide21 (23 mg, quant.) as a colorless powder. R_(f) 0.3 (100:10:10:10:2(v/v/v/v/v) ethanol-n-butanol-pyridine-water-acetic acid), [α]_(D) +42°(c 1; water), m/z 584.9 (M⁺+Na).

¹H NMR, D₂O: 1.98-2.05 (m, 2H, —CH ₂—), 3.17 (m, 2H, —CH ₂NH₂),3.33-3.35 (m, 1H, H-2a), 4.36 (m, 1H, H-5c), 4.53 (d, 2H, J=7.8, H-1a,H-1b), 4.97 (d, 1H, J_(1,2)=3.67, H-1c).

Preparation of 2-azidoethyl(3,4-di-O-acetyl-2,6-di-O-benzyl-α-D-galactopyranosyl)-(1→4)-(2,3,6-tri-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside(25)

To the solution of ethyl3,4-di-O-acetyl-2,6-di-O-benzyl-1-thio-β-D-galactopyranoside (23) (550mg, 1.11 mmol) in dichloromethane (10 ml) was added Br₂ (57 μl, 1.11mmol). The mixture was held for 20 min at room temperature, thenconcentrated in vacuo at room temperature and co-evaporated withanhydrous benzene (3×30 ml). The crude3,4-di-O-acetyl-2,6-di-O-benzyl-α-D-galactopyranosylbromide (24) wasused for glycosylation without purification.

The mixture of lactose derivative 22 (Sun et al (2006)) (500 mg, 0.525mmol), 1,1,3,3-tetramethylurea (300 μl), molecular sieves MS 4 Å (1 g),and dichloromethane (25 ml) was stirred for 30 min at room temperature.Silver trifluoromethanesulfonate (285 mg, 1.11 mmol), molecular sievesMS 4 Å (0.5 g), and the freshly prepared galactopyranosylbromide (24) indichloromethane (15 ml) were then added. The reaction mixture wasstirred overnight, filtered, and concentrated in vacuo.

Column chromatography on Silica gel (elution with 3:1 to 1:1 (v/v)hexane-ethyl acetate) resulted in 570 mg (79%) of trisaccharide 25,R_(f) 0.25 (2:1 (v/v) hexane-ethyl acetate); [α]_(D) +32° (c 0.8, CHCl₃)

¹H NMR, CDCl₃: 1.88, 1.94 (2s, 2Ac), 3.00 (dd, 1H, J_(5,6)=4.9,J_(6′,6)=8.4, H-6a), 3.19 (dd, J_(1,2)=8.5, J_(2,3)=8.9, H-2a),3.30-3.36 (m, 2H, —CHHN₃, H-6′a), 3.38-3.47 (m, 4H, H-5a, H-5b, H-2b,H-6b), 3.48-3.54 (m, 1H, —CHHN₃), 3.61 (dd, 1H, J_(2,3)=8.9,J_(3,4)=9.2, H-3a), 3.69-3.75 (m, 3H, H-6′b, H-6c, —OCHH—), 3.85 (dd,1H, J_(5,6)=4.6, J_(6,6′)=11.0, H-6c), 3.89 (dd, 1H, J_(1,2)=3.4,J_(2,3)=10.8, H-2c), 3.95 (dd, 1H, J_(3,4)=9.2, J_(4,5)=9.5, H-4a),4.0-4.1 (m, 4H, —OCHH—, H-4b, CH₂Ph), 4.25, 4.29, 4.32, 4.39 (4 d, 4×1H,J_(AB)=12, 4 —CHPh), 4.43 (d, 1H, J_(1,2)=7.6, H-1), 4.48 (d, 1H,J_(1,2)=7.6, H-1), 4.54-4.62 (m, 5H, 4 —CHPh, H-5c), 4.71-4.84 (m, 4H, 4—CHPh), 4.89, 4.91, and 5.09 (3 d, 3=1H, 3 4 —CHPh), 5.15 (d, 1H,J_(1,2)=3.0, H-1c), 5.39 (dd, 1H, J_(2,3)=10.8, J_(3,4)=3.4, H-3c), 5.56(dd, 1H, J_(3,4)=3.4, J_(4,5)=0.9, H-4c), 7.14-7.48 (m, 40H, Ph).

Preparation of 2-aminoethyl α-D-galactopyranosyl-(1→4)-β-D-galactopyranosyl-(1→4) -β-D-glucopyranoside (Gb₃-S₁) (28)

Sodium methylate (100 μl of 2 M solution in methanol) was added to asuspension of trisaccharide (25) (500 mg, 0.363 mmol) in anhydrousmethanol (50 ml). The mixture was stirred overnight at room temperature,quenched with acetic acid, and concentrated in vacuo.

Column chromatography on Silica gel (elution with 2:1 to 1:1 (v/v)hexane-ethyl acetate) resulted in 470 mg of trisaccharide (26), R_(f)0.5 (1:1 (v/v) hexane-ethyl acetate), [α]_(D) +36° (c 0.5, CHCl₃).

To a solution of trisaccharide (26) and Boc₂O ((150 mg, 0.91 mmol) inanhydrous methanol (50 ml) was added the catalyst 10% Pd/C (500 mg). Themixture was degassed and the flask filled with hydrogen. The reactionmixture was stirred for 3 h, filtered off from the Pd/C, andconcentrated in vacuo.

Column chromatography on Silica gel (elution with 6:5:1 (v/v/v)chloroform-ethanol-water) resulted in 160 mg (68%) of trisaccharide 27R_(f) 0.3 (6:5:1 (v/v/v) dichloromethane-ethanol-water). ¹H NMR, D₂O:1.45 (s, 9H, (CH₃)₃COCO—), 4.53 (d, 1H, J_(1,2)=7.8, H-1b), 4.58 (d, 1H,J_(1,2)=7.4, H-1b), 4.98 (d, 1H, J_(1,2)=3.0, H-1c).

The trisaccharide 27 was then treated with 95% CF₃COOH (5 ml, 10 min).Upon completion, the mixture was concentrated in vacuo, co-evaporatedwith toluene, and applied to a column (10×100 mm) of Dowex 50X4-400 (H⁺)cation exchange resin. The target compound was eluted with 1 M aqueousammonia and the eluant was concentrated in vacuo. Lyophilization fromwater provided trisaccharide 28 (135, quant.) as a colorless powder.R_(f) 0.35 (100:10:10:10:2 (v/v/v/v/v)ethanol-n-butanol-pyridine-water-acetic acid), [α]_(D) +25° (c 0.2;water).

¹H NMR, D₂O: 3.32 (m, 2H, —CH₂—NH₂), 3.40-3.45 (m, 1H, H-2a), 3.63 (dd,1H, J_(1,2)=7.9, J_(2,3)=10.3, H-2b), 3.66-3.78 (m, 5H, H-5a, H-3a,H-4a, H-6c, H-6′c), 3.8 (dd, 1H, J_(3,4)=3.1, J_(3,2)=10.3, H-3b), 3.84(m, 2H, J_(5,6)=4.4, J_(5,6′)=7.9, H-5b), 3.88-3.92 (m, 3H, H-2c, H-6b,—OCHH—), 3.96 (dd, 1H, J_(3,4)=3.3, J_(3,2)=10.3, H-3c), 3.98-4.03 (m,2H, H-6a, H-6′b), 4.06 (dd, 1H, J_(5,6)=2.2, J_(6,6′)=12.3, H-6′a), 4.08(dd, 1H, J_(3,4)=3.3, J_(4,5)=0.9, H-4c), 4.09 (d, 1H, J_(3,4)=3.1,H-4b), 4.17-4.21 (m, 1H, —OCHH—), 4.41 (m, 1H, H-5c), 4.56 (d, 1H,J=7.9, H-1b), 4.60 (d, 1H, J=8.1, H-1a), 5.00 (d, 1H, J_(1,2)=3.9,H-1c).

The preparation of the primary amino propyl glycosidesGalNAcα1-3(Fucα1-2)Galβ-O(CH₂)₃NH₂ (A_(tri)r-S₁) andGalα1-3(Fucα1-2)Galβ-O(CH₂)₃NH₂ (B_(tri)-S₁) is described in thepublication of Korchagina and Bovin (1992). The preparation of theprimary amino propyl glycosides Xylα1-3Glcβ-O(CH₂)₃NH₂ andXylα1-3Xylα1-3Glcβ-O(CH₂)₃NH₂ is described in the publication of Krylovet al (2007). The preparation of the primary amino propyl glycosidesNeu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ-O(CH₂)₃NH₂ (sLe^(a)-S₁) andNeu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ-O(CH₂)₃NH₂ (sLe^(x)-S₁) is describedin the publication of Nifant'ev et al (1996). The preparation of theprimary amino propyl glycosidesGalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ-O(CH₂)₃NH₂ (A_(tetra)(Type 2)-S₁),Galα1-3(Fucα1-2)Galβ1-4GlcNAcβ-O(CH₂)₃NH₂ (B_(tetra)(Type 2)-S₁)Fucα1-2Galβ1-4GlcNAcβ-O(CH₂)₃NH₂ (H_(tri) (Type 2)-S₁),Galβ1-4(Fucα1-3)GlcNAcβ-O(CH₂)₃NH₂ (Le^(x)-S₁),Fucα1-2Galβ1-4(Fucα1-3)GlcNAcβ-O(CH₂)₃NH₂ (Le^(y)-S₁),Galα1-3Galβ1-4GlcNAcβ-O(CH₂)₃NH₂ (B_(tri) (Type 2)-S₁) andGalα1-4Galβ1-4GlcNAcβ-O(CH₂)₃NH₂ (P₁-S₁) is described in the publicationof Pazynina et al (2002). The preparation of the primary amino propylglycosides Neu5Acα2-3Galβ-O(CH₂)₃NH₂,Neu5Acα2-3Galβ1-4GlcNAcβ-O(CH₂)₃NH₂ (3′SLN-S₁),Neu5Acα2-3Galβ1-4(6-HSO₃)GlcNAcβ-O(CH₂)₃NH₂ (6-Su-3′SLN-S₁),Neu5Acα2-3Galβ1-3GalNAcα-O(CH₂)₃NH₂ (SiaTF-S₁),Neu5Acα2-3Galβ1-3(6-HSO₃)GalNAcα-O(CH₂)₃NH₂ (6-Su-SiaTF-S₁),Neu5Acα2-3Galβ1-3GlcNAcβ-O(CH₂)₃NH₂ (SiaLe^(c)-S₁),Neu5Acα2-3Galβ1-3(6-HSO₃)GlcNAcβ-O(CH₂)₃NH₂ (6-Su-SiaLe^(c)-S₁),Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ-O(CH₂)₃NH₂ (SiaLe^(a)-S₁),Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ-O(CH₂)₃NH₂ (SiaLe^(x)-S₁) is describedin the publication of Pazynina et al (2003). The preparation of theprimary amino propyl glycosideGalβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-O(CH₂)₃NH₂(trilactosamine-S₁) is described in the publication of Pazynina et al(2008). The preparation of the primary amino propyl glycosidesNeu5Ac-α-(2-6′)-Galβ1-4GlcNAcβ-O(CH₂)₃NH₂(Neu5Ac-α-(2-6′)-lactosamine-S₁) andNeu5Gc-α-(2-6′)-Galβ1-4GlcNAcβ-O(CH₂)₃NH₂(Neu5Gc-α-(2-6′)-lactosamine-S₁) is described in the publication ofSherman et al (2001).

Preparation of Galili-CMG(2)-DOPE (22)

To a stirred solution of compound 21 (66 mg, 0.079 mmol) in dry DMSO (6mL) were added 15 μl Et₃N and powdered H₂N-CMG(2)-DOPE (20) (95 mg,0.0495 mmol) in 3 portions. The mixture was stirred for 24 h at roomtemperature and then subjected to column chromatography (Sephadex LH-20,i-PrOH—H₂O, 1:2, 0.5 v % Py, 0.25 v % AcOH) to yield the crude compound22 in a form of Py-salt; The compound was lyophilized from water twotimes, then dissolved again in 10 ml of water, aqueous solution ofNaHCO₃ (50 mM) was added to pH 6.5 for obtaining the compound 22 in aform of Na-salt and the solution was subjected to lyophilization. Theyield of compound 22 (Na-salt) was 114 mg (86% based on NH₂-CMG₂-DE),R_(f) 0.6 (i-PrOH-MeOH-MeCN—H₂O, 4:3:6:4). ¹H NMR (FIG. 4) (700 MHz,D₂O-CD₃OD, 1:1 (v/v), 40° C.; selected signals) δ, ppm: 1.05 (t, J 7.03Hz, 6H; 2 CH ₃), 1.40-1.58 (m, 40H; 20 CH ₂), 1.73-1.87 (m, 12H; 2×—COCH₂CH ₂CH ₂CH₂CO and 2× —COCH₂CH ₂—), 1.90-1.99 (m, 2H; OCH₂CH₂CH₂N), 2.15-2.25 (m, 11H; 2× —CH ₂CH═CHCH ₂—, NHC(O)CH ₃), 2.39-2.59(2m, total 12H, 2× —COCH₂CH ₂CH ₂CH₂CO— and 2× —COCH₂CH ₂—) 4.63 (dd,1H, J 2.51, J 12.20, C(O)OCHHCHOCH₂O—), 4.67 and 4.69 (2d×1H, J_(1,2)7.81, J_(1,2) 7.95, H-1^(I), H-1^(II)), 5.30 (d, 1H, J_(1,2) 3.88,H-1^(III)), 5.42-5.46 (m, 1H, —OCH₂—CHO—CH₂O—), 5.49-5.59 (m, 4H, 2×—CH═CH—); MALDI TOF mass-spectrum, M/Z: 2567 (M+Na); 2583 (M+K); 2589(MNa+Na); 2605 (MNa+K); 2611 (MNa₂+Na).

Biology

The use of the peptide-lipid constructs in methods for effectingqualitative and quantitative changes in the levels of peptide expressedat the surface of cells and multi-cellular structures was illustratedwith reference to serodiagnosis. In the following table cross-reactivityof polyclonal sera and monoclonal antibodies of known specificities andred blood cells (RBCs) modified with the constructDOPE-Ad-CMG(I)-βAla-Mal-Mur(D14C) (XI) (2 hours, 37° C.) is summarized.

EIA/Miltenberger Reagent ID Type Specificity 2 T217 Human group AB serumReactive with MUT-T peptides by EIA 3 T165 Human group O serum Reactivewith MUR peptides by EIA 4 T7202 Human group B serum Reactive with MUT-Mpeptides by EIA 6 T6025 Human group A serum Reactive with MUT-T peptidesby EIA 7 T8445 Human group O serum Uncertain 8 T5896 Human group O serumUncertain 9 MIII Monoclonal antibody Reactive with Mi III red cells 10Mia Monoclonal antibody Reactive with Mi III red cells 11 Mur Monoclonalantibody Reactive with Mur positive red cells 12 Gam IgG monoclonalReactive with Mi III red antibody cells 13 BoxH Human serum Uncertain 14TAP1 Human group O serum Presumed MUT-K specificity 15 TAP2 Human serumPresumed MUR specificity

Trans- Untrans- Anti- formed formed body ID Specificities cells cellsExpected T165 serum Mur 10 0 postives T6025 serum K + Mur 5 0 T8445serum Mur + Hil + 5 0 Tsen T5896 serum M + K + 0 0 (Mur) Expected JapanMoAb Mur 0 0 negatives T4130 serum Hil + Tsen 0 0 T217 serum T 0 0 T7202serum M 0 0 T8012 serum M + K + T 0 0 Japan MoAb Mi III (1:10) 0 0 Boxserum ? 0 0 Hill Japan MoAb Mi^(a) (1:50) 0 0 E119 KBL MoAb Mia 0 0 7201GAMMA(1:100)Modification of Red Blood Cells with Peptide-Lipid Constructs

Red blood cells are modified by mixing 1 part by volume of washed packedred blood cells with 1 part by volume of peptide-lipid constructdispersed at a concentration of 10 to 1000 μg/ml in cell media(Celpresol™).

The suspensions are either:

-   -   1. incubated for 2 hours at 37° C. before being washed and        suspended in a cell medium for serological analysis at a        concentration of 0.8 to 3% (Method 1); or    -   2. incubated for 3 to 4 hours at room temperature (circa 25° C.)        followed by 18 hours at 4° C. before being washed and suspended        in a cell medium for serological analysis at a concentration of        0.8 to 3% (Method 2).

Tube Serology Testing of Modified Red Blood Cells

Serological reactions are graded or scored by either of two establishedsystems (0 or ‘-’=no agglutination, 1+ or 3=very weak agglutination, 2+or 5=weak agglutination, 3+ or 8=moderate strong agglutination, 4+ or10/12=strong agglutination)

Serological platforms used are Tube (addition of reagents and reactantsinto plastic or glass serology tubes and after appropriate incubations,washing and centrifugation observing reactions macroscopically by eyeand a 10× magnification eyepiece and scoring) and BioVue™ (addition ofreactants into cassettes containing beads (including some reactants) andafter appropriate incubations and centrifugation observing the reactionpatterns trapped within the Gel matrix). BioVue is the serologicalcolumn agglutination platform of Ortho-Clinical Diagnostics. Diamed isthe serological column agglutination platform of Diamed AG.

Serum samples were available from 47 blood donors of negative antibodyscreen status. These samples were designated “negative samples”, but notdetermined not to have anti-Miltenberger antibodies).

Three serum samples known to have Miltenberger related antibodies T217,T6025, T5896. These samples were designated “positive samples”, but notdetermined to have anti-antibodies against the peptide of the peptide ofthe construct designated DOPE-PEG₆-βAla-Mal-Milt(K) (M00).

A suspension of 3% modified RBCs was prepared in PBS and 30 μl of thesuspension mixed with 30 μl serum sample. The mixtures were thenincubated for 45 min at 37° C. Following incubation the RBCs werecentrifuged for 10 s in an Immufuge™ (setting: “high”) and observed foragglutination before being washed 3 times with PBS.

After washing one drop of Epiclone™ anti-human globulin (AHG) was addedand the tubes then centrifuged for 10 s in an Immufuge™ (setting:“high”). Tubes were then read and serology scores recorded.

Comments on the observed serology scores are provided in the legends tothe following tables.

TABLE 1 Summary of reactivity of samples of serum from 47 blood donorsnot expected to have anti-Miltenberger activity (“negative samples”).AHG+ means sample reacted by the anti-human globulin test. AHG− meanssample is unreactive. RBCs were modified with the peptide-lipidconstruct designated DOPE-PEG₆-βAla-Mal-Milt(K) at the concentrationsindicated. Sera were tested against modified RBCs following 3 daysstorage. Age of Concentration of DOPE-PEG₆-βAla- modifiedMal-Milt(K)(M00) (mg/ml) RBCs 1.0 0.5 0.25 (days) Serum (n = 47) (n =21) (n = 21) 3 Negative AHG+ AHG− AHG+ AHG− AHG+ AHG− samples 1 46 0 210 21

TABLE 2 Results by tube serology of 3 serums known to contain antibodiesagainst antigens of the Miltenberger complex. Score results show samplereactivity by the anti-human globulin test, 1+ = weak, 2+ = medium, 3+ =medium/strong, 4+ = strong, — means sample is unreactive. RBCs weremodified with the peptide-lipid construct at the concentrationsindicated. Sera were tested against modified RBCs following 3 days and24 days storage. (n.t.—not tested). Age of Concentration ofDOPE-PEG₆-βAla- modified Mal-Milt(K)(M00) (mg/ml) RBCs (days) Serum 1.00.5 0.25 3 T217 2+ 1+ — 3 T6025 4+ 4+ 4+ 3 T5896 — — — 24 T217 — — n.t.24 T6025 2+ 2+ n.t. 24 T5896 — — n.t.

TABLE 3 Results by Diamed column serology of 3 serums known to containantibodies against the Miltenberger complex. Score results show samplereactivity by the anti-human globulin test, 1+ = weak, 2+ = medium, 3+ =medium/strong, 4+ = strong, — means sample is unreactive. RBCs weremodified with the peptide-lipid construct at the concentrationsindicated. Sera were tested against modified RBCs following 3 days and24 days storage. Age of Concentration of DOPE-PEG₆-βAla- modifiedMal-Milt(K)(M00) (mg/ml) RBCs (days) Serum 1.0 0.5 0.25 3 T217 — — 1+ 3T6025 1+ 2+ 1+ 3 T5896 — — — 24 T217 — — — 24 T6025 2+ 2+ 1+ 24 T5896 —— —

TABLE 6 Identification of naturally occurring Miltenberger antigenpositive (Milt⁺) human red cells as determined in BioVue AHG cards.Polyclonal sera Monoclonal antibodies 2 3 4 6 7 8 14 15 9 10 11 12 CellID Antigen T217 T165 T7202 T6025 T8445 T5896 TAP1 TAP2 MIII Mia Mur Gam9422184 Vw 8 5 3 0 8 0 5 0 0 10 0 12 11297161 MiIII 12 10 12 12 10 10 1010 10 12 12 4131850 MiIV 12 12 10 0 10 12 12 1523 MiVI 12 12 8 0 10 1210 T1569 MiVII 0 0 0 0 10 0 0 0 0 0 0 C.BR Mi?X 12 10 12 12 8 12 12 8 010 10 10

TABLE 7 Identification of peptide-lipid constructs. Lowercase‘c’ denotes a cysteine residue (Cys). All peptide Lipid constructs(F-S-L or L-S-F) were prepared as the DOPE (L) variant. M refers to ashorthand name for the molecule construct and is used in the followingtables. The terminal peptide sequence is as indicated with. “little c”representing Cys via which S is linked to L. Spacer refers to thestructural motif of the spacer (S). CMG denotes the peptide-lipidconstructs described in this specification. PEG denotes peptide-lipidconstructs of the structure described as the second aspect of theinvention in the specification accompanying the international PCTapplication filed on 11 Sep. 2008 at the Intellectual Property Office ofNew Zealand as receiving Office (RO/NZ). All constructs were prepared asthe DOPE variant. Peptide sequence Leader spacer 13 MUTK Ser Ser Gln ThrAsn Asp Lys His Lys Arg Asp Thr Tyr c PEG6 34 MUTK Ser Ser Gln Thr AsnAsp Lys His Lys Arg c CMG(2) 21 MUTK     Ser Gln Thr Asn Asp Lys His LysArg Asp c CMG(2) 22 MUTK     Ser Gln Thr Asn Asp Lys His Lys Arg Asp cCMG(2) 36 MUTK     Ser Gln Thr Asn Asp Lys His Lys Arg Asp Thr c CMG(2)35 MUTK         Gln Thr Asn Asp Lys His Lys Arg Asp Thr c CMG(2) 1 MUTK        Gln Thr Asn Asp Lys His Lys Arg Asp Thr Tyr AAAAA PEG6 2 MUTK        Gln Thr Asn Asp Lys His Lys Arg Asp Thr Tyr GSerGSerGc PEG6 3MUTK         Gln Thr Asn Asp Met His Lys Arg Asp Thr Tyr GSerGSerGc PEG69 MUTK         Gln Thr Asn Asp Lys His Lys Arg Asp Thr Tyr GSerGSerGcCMG(2) 33 MUTK         Gln Thr Asn Asp Lys His Lys Arg Asp Thr Tyr cCMG(2) 37 MUTK             Thr Asn Asp Lys His Lys Arg Asp Thr Tyr Pro cCMG(2) 14 Mur Asp Thr Tyr Pro Ala His Thr Ala Asn Glu Val Ser Glu cCMG(2) 14 Mur Asp Thr Tyr Pro Ala His Thr Ala Asn Glu Val Ser Glu cCMG(2) 14 Mur Asp Thr Tyr Pro Ala His Thr Ala Asn Glu Val Ser Glu cCMG(2) 30 Mur Asp Thr Tyr Pro Ala His Thr Ala Asn Glu c CMG(2) 16 Mur    Thr Tyr Pro Ala His Thr Ala Asn Glu Val c PEG 17 Mur     Thr Tyr ProAla His Thr Ala Asn Glu Val c CMG(2) 28 Mur     Thr Tyr Pro Ala His ThrAla Asn Glu c CMG(2) 27 Mur     Thr Tyr Pro Ala His Thr Ala Asn c CMG(2)25 Mur         Tyr Pro Ala His Thr Ala Asn Gln c CMG(2) 26 Mur        Tyr Pro Ala His Thr Ala Asn Glu Val c CMG(2) 31 Mur         TyrPro Ala His Thr Ala Asn Glu Val Ser c CMG(2) 18 Mur             Pro AlaHis Thr Ala Asn Glu Val c CMG(2) 19 Mur             Pro Ala His Thr AlaAsn Glu Val c CMG(2) 29 Mur             Pro Ala His Thr Ala Asn Glu ValSer c CMG(2) 40 Hil Glu Glu Glu Thr Gly Glu Thr Gly Gln Leu c CMG(2) 23Hil     Glu Glu Thr Gly Glu Thr Gly Gln Leu Val c CMG(2) 24 Hil     GluGlu Thr Gly Glu Thr Gly Gln Leu Val c CMG(2) 41 Hil         Glu Thr GlyGlu Thr Gly Gln Leu Val His c CMG(2)

TABLE 8 Analysis of sorted data for the reactivity against theMiltenberger Antibody Positive Panel of RBCs modified to incorporate theMUT peptide-lipid constructs identified at the concentration indicated.Constructs were able to show reactivity with one or more polyclonalserums indicating specificity to one or more peptide variations.Miltenberger Antibody Positive Panel 4 8 2 6 3 14 7 9 10 11 12 13 15 Mμg/ml T7202 T5896 T217 T6025 T165 TAP1 T8445 MIII Mia Mur Gam BoxH TAP213 250 8 3 8 8 0 0 0 0 0 0 8 34 50 0 0 0 0 3 0 21 200 0 0 0 8 8 0 0 0 03 5 22 200 0 0 0 10 0 0 0 0 0 3 0 36 50 0 0 0 0 8 0 35 50 0 0 0 0 5 0 1500 5 0 3 8 0 0 0 5 0 8 2 500 8 8 8 8 5 0 0 5 0 8 9 300 8 10 8 8 8 3 0 00 8 10 33 50 0 0 0 0 8 0 37 50 8 0 5 0 8 0 3 1000 8 10 0 5 0 0 0 0 5

TABLE 9 Analysis of sorted data for the reactivity against theMiltenberger Antibody Positive Panel of RBCs modified to incorporate theMUR peptide-lipid constructs identified at the concentration indicated.Constructs were able to show reactivity with one or more polyclonalserums indicating specificity to one or more peptide variations.Miltenberger Antibody Positive Panel 3 6 7 4 8 2 15 9 10 11 12 13 Mμg/ml T165 T6025 T8445 T7202 T5896 T217 TAP2 MIII Mia Mur Gam BoxH 14 1010 8 5 0 0 0 0 0 0 0 14 50 10 5 8 3 0 0 0 0 0 14 100 10 10 5 5 0 3 0 0 00 30 50 8 10 0 0 8 16 100 10 5 12 5 0 0 0 0 0 0 0 17 100 10 10 10 8 0 00 0 0 0 0 28 50 8 10 0 0 8 27 50 0 10 0 0 0 25 50 3 0 3 0 0 0 8 0 0 0 026 50 10 8 8 0 0 0 3 0 0 0 0 31 50 8 10 0 0 0 18 100 10 10 8 0 0 0 0 0 00 19 100 10 8 10 0 3 0 0 0 0 0 29 50 10 0 0 8

TABLE 10 Negative serum reactivity. Miltenberger negative red cells weremodified with the peptide- lipid construct M22 at a transformationconcentration of 50 μg/ml and tested against antibody negative serums inthe field to determine rates of false positivity. Studies wereundertaken in clinical laboratories in Australia, Malaysia andPhilippines using three different serological platforms; Columnagglutination platforms BioVue and DiaMed as well as the simpletechnique of tube reactivity. Equal volumes of packed RBCs and asolution containing 50 μg/ml of the construct were contacted for 3 hoursat room temperature and then 18 hours at 4° C. This field trial foundthat clinical antibody negative serums reacted with M22 transformedcells at rates 0.4 to 4.5% in the BioVue ™ platform and at a rate of0.4% in the DiaMed ™ platform. No reactivity was observed in the tubeplatform. These results can be considered as false positive reactions.Number Tested Number Positive % Positive Country Laboratory BioVueDiaMed Tube BioVue DiaMed Tube BioVue DiaMed Tube Australia CSL 100 33.0 Melb Path 45 2 4.5 RNSH 500 500 2 2 0.4 0.4 Malaysia UMMC 749 19 2.5Philippines Metrop Hosp 60 0 0

TABLE 11 Positive serum reactivity. Miltenberger negative red cells weremodified with the peptide-lipid construct M22 at a transformationconcentration of 50 μg/ml, M17 at a transformation concentration of 200μg/ml, M24 at a transformation concentration of 200 μg/ml, and testedagainst natural Mi III antibody reactive human serums in the field todetermine rates of reactivity. Equal volumes of packed RBCs and asolution containing 50 μg/ml of the construct were contacted for 3 hoursat room temperature and then 18 hours at 4° C. The three differentconstructs of MUT, MUR and HIL were able to discriminate most natural MiIII reactive polyclonal antibodies into specific reactivity profiles.Twelve serums were unreactive with the modified cells, suggesting theymay have specificity against another Mi III antigen. 22 (50 17 (200 24(200 Sample No. μg/ml) μg/ml) μg/ml) Interpretation 488-6 10 K9327986660 8 3 0 K 9325490091 5 5 0 K 9328791834 5 3 0 K 621-3 5 K922390844-5 0 12 0 Mur 9322338631 0 10 0 Mur 914146821-8 0 10 0 Mur932809044-1 0 8 0 Mur 942433813-3 0 8 0 Mur 942404708-4 0 5 0 Mur942421413-0 0 5 0 Mur 942223755-1 0 5 0 Mur 942442720-2 0 5 0 Mur927619701-8 0 3 0 Mur 912485657-9 0 3 0 Mur 926190919-0 0 3 0 Mur9328154853 0 10 3 Mur + Hil 9328118428 0 10 5 Mur + Hil 9425256505 0 8 8Mur + Hil 942433855-3 0 8 8 Mur + Hil 942753165-4 0 8 8 Mur + Hil9424292604 0 8 5 Mur + Hil 9427455417 0 5 5 Mur + Hil S-3 0 3 5 Mur +Hil 942448627-8 0 0 5 Hil 942423002-4 0 0 3 Hil 942762589-1 0 0 3 Hil9424248012 0 0 0 other 9427615156 0 0 0 other 9424396133 0 0 0 other9427613497 0 0 0 other 927175131-4 0 0 0 other 932467774-5 0 0 0 other927299700-1 0 0 0 other 926555294-1 0 0 0 other 932360876-4 0 0 0 other927516053-2 0 0 0 other 942404708-4 0 0 0 other 589-6 0 other

TABLE 12 Anti-MUT serum reactivity. Miltenberger negative RBCs weremodified with the peptide-lipid constructs M22 at concentrations rangingfrom 10 to 200 μg/ml, M17 at a concentration of 50 μg/ml and M24 at aconcentration of 50 μg/ml. The modified cells were tested against anatural Mi III antibody reactive Taiwan human serum (TAP1) detected inthe field to determine reactivity profile. Reactivity was comparedagainst natural Mi III antigen positive cells. TAP1 (Taiwan Miltenbergerantibody positive sample). TAP1 serum was shown to contain both IgG andIgM antibodies (the latter being saline reactive) directed againstnatural MiIII positive cells. The lack of reactivity againstAbtectcell ™ and Phenocell ™ antibody screening and identificationpanels concludes no other antibodies against red cells are present.Reactivity with M22 modiifed cells over transformation concentrations of10 to 200 μg/ml (and not with untransformed cells - 0 μg/ml) concludesthe presence of an antibody directed against MUT. The failure of the M22modified cells to detect a saline reaction, suggests the assay issensitive to the IgG class of antibody and not IgM - a clinicallyfavourable result. The lack of reactivity with the M17 and M24transformed cells concludes and absence of antibodies to the MUR and HILmutations. TAP 1 Serum RBCs Saline AHG Natural Mi III positive cellsR2R2, 11297161 8 10 R1Rz 11291347 8 10 Abtectcell III 8245009 10Peptide-lipid Construct (μg/ml) Modified cells M22 (MUT) 200 0 10 M22(MUT) 100 0 8 M22 (MUT) 50 0 8 M22 (MUT) 20 0 8 M22 (MUT) 10 0 5 M22(MUT) 0 0 0 M17 (Mur) 0 0 M24 (Hil) 0 0 Antibody Screen/ID panel 0Abtectcell III Batch 2223005 Cells I-III Phenocell B Batch 2653046 0Cells 1-11

TABLE 13 Anti-MUT serum reactivity. Miltenberger negative red cells weretransformed with the peptide-lipid constructs M28 at a concentration of100 μg/ml and M22 at a concentration of 100 μg/ml and tested against anatural Mi III antibody reactive Taiwan human serum (TAP2) detected inthe field to determine reactivity profile. Reactivity was comparedagainst natural Mi III antigen positive cells. TAP 2 (TaiwanMiltenberger antibody positive sample). TAP2 serum was shown to containIgG antibodies antibodies directed against natural Mi III positivecells. The lack of reactivity against Abtectcell ™ antibody screeningand identification panels concludes no other antibodies against redcells are present. Reactivity with M28 modified cells and not with M22modiifed cells concludes the presence of an antibody directed againstthe MUR peptide. TAP 2 Serum RED CELLS BioVue AHG Natural Mi IIIpositive cells Mi III PDN No. 54 5 Mi IV No; 4131850 10 Mi VI 1523A 8 Mi?x ID: CBR 8 Vw No: 9422184 0 Peptide-lipid construct (μg/ml) M28 (MUR)100 10 M22 (MUT) 100 0 Antibody Screen panel Abtectcell III Batch2223009 Cells I-III 0

TABLE 14 Identification of the M37 sequence as a candidate for thedetection of anti-MUT. Miltenberger negative cells were modiifed withthe peptide-lipid constructs M22, M33, M34, M35, M36, M37, M40 and M41 aconcentration of 50 μg/ml. Modified cells were tested against serums 2,3, 4 and 8 of the Miltenberger Antibody Positive Panel and Taiwan Mi IIIantibody positive serum TAP1 to determine its MUT reactivity profile.TAP 1 (Taiwan Miltenberger antibody positive sample). Cells modifiedwith the peptide-lipid construct M37 were able to detect the anti-MUTactivity of Miltenberger Antibody Positive Panel samples 2 and 4. Sample3 containing MUR activity was expected negative. Sample 8 containingmultiple antibodies was unexpectedly negative, but may have lostspecificity. TAP1 serum was able to detect all MUT variations,indicating some polyclonal serums may have less defined anti-MUTactivity than others. BioVue Positive Sample ID Specificities M22 M33M34 M35 M36 M37 M40 M41 Unmodified 3 T165 Mur 0 0 0 0 0 0 0 0 0 8 T5896M + K + Mur 0 0 0 0 0 0 0 0 0 2 T217 T 0 0 0 0 0 5 0 0 0 4 T7202 M 0 0 00 0 8 0 0 0 TAP1 8 3 5 5 8 8 0 0 0 MUT MUT/MUR MUR MUT peptides #2 #4TAP1 #8 #3 34 MUTK4 S S Q T N D K H K R − − ++ − − 22 MUTK3 S Q T N D KH K R D − − +++ − − 36 MUTK6 S Q T N D K H K R D T − − +++ − − 35 MUTK5Q T N D K H K R D T − − ++ − − 33 MUTK1 Q T N D K H K R D T − − + − − 37MUTK7 T N D K H K R D T ++ +++ +++ − − MUT MUT/MUR MUR HIL peptides #2#4 TAP1 #2 #4 40 Hil 2 E E E T G E T G Q L − − − − − 41 Hil 3 E T G E TG Q L V H − − − − −

TABLE 15 Anti-MUT serum (TAP1) reactivity. Miltenberger negative cellswere modified with the peptide-lipid constructs M22, M33, M34, M35, M36,M37, M40 and M41 at a concentration of 50 μg/ml (2 hours, 37° C.).Modified cells were tested against Taiwan Mi III antibody positive serumTAP1 to determine its MUT reactivity profile. TAP 1 (Taiwan Miltenbergerantibody positive sample). The TAP1 serum is able to recognize some, butnot all, peptide variations of MUT. The lack of reactivity with M40 andM41 (HIL peptide) modified cells and the untransformed cells isexpected. Reactivity MUT Peptides 34 MUTK4 S S Q T N D K H K R 5 22MUTK3 S Q T N D K H K R D 8 36 MUTK6 S Q T N D K H K R D T 8 35 MUTK5 QT N D K H K R D T 5 33 MUTK1 Q T N D K H K R D T 3 37 MUTK7 T N D K H KR D T 8 HIL peptides 40 Hil 2 E E E T G E T G Q L — 41 Hil 3 E T G E T GQ L V H —

TABLE 16 Anti-MUR serum (TAP1) reactivity. Miltenberger negative cellswere modified with the peptide-lipid constructs M17, M19, M25, M26, M27,M28, M29, M30 and M31 at a concentration of 50 μg/ml (2 hours, 37° C.).Modified cells were tested against antibody positive serum #7 from theMiltenberger Antibody Positive Panel to determine their MUR reactivityprofile. Panel Serum #7 (T8445) 30 Mur 9 D T Y P A H T A N E 8 17 Mur 2T Y P A H T A N E V 10 28 Mur 6 T Y P A H T A N E 8 27 Mur 5 T Y P A H TA N — 25 Mur 4 Y P A H T A N E 3 26 Mur 7 Y P A H T A N E V 10 31 Mur 10Y P A H T A N E V S 8 19 Mur 3 P A H T A N E V 8 29 Mur 8 P A H T A N EV S 8

TABLE 17 False positive MUT construct reactions with negative serums.The rate of false positive reactions was determined against a panel of51 blood donor plasma samples (PAC1-51). Plasma were tested againstcells modified with the peptide- lipid constructs M22, M34, M36, M37 andM40 of peptide-lipid constructs at a concentration of 50 μg/ml (2 hours,37° C.) and tested in BioVue AHG cards. The amino acid sequence caninfluence the rate of false positive reactions. One more or less aminoacid at either end of the polypeptide chain can increase the chances ofnon-specific reactions occurring with serum. Reaction scores - BioVueAHG PAC Samples No. M22 M34 M36 M37 M40 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 00 0 0 0 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 49, 50, 52, 53 47 0 0 3 0 0 48 0 3 5 0 0 51 0 5 0 0 0 % FalsePositives 0 3.8% 3.8% 0 0 Reactivity MUT Peptides 34 MUTK4 S S Q T N D KH K R 3.8% 22 MUTK3 S Q T N D K H K R D 0 36 MUTK6 S Q T N D K H K R D T3.8% 37 MUTK7 T N D K H K R D T 0 HIL peptides 40 Hil 2 E E E T G E T GQ L 0

TABLE 18 False positive MUR M17 construct reactions with 102 negativeserums. Cells modified with the peptide-lipid construct M17 were testedagainst 102 negative serum samples. Cells modified with thepeptide-lipid construct M17 give the most “false positive” reactiveconstruct showing up to 36% false positive rate with negative serums.False positive reactions with M17 Score 12-10 8-5 3  0 (n = 102) 17 18 365 17% 18% 3% 64%

TABLE 19 “M17-false-positive” negative serum reactivity against otherMur constructs. The 6 most false positive negative serums reactiveagainst cells modified with the peptide-lipid construct M17 were testedagainst cells modified by contacting with the peptide-lipid constructsM19, M25, M26, M27, M28, M29, M30 and M31 at a concentration of 50 μg/ml(2 hour 37° C.). The modified cells were tested in BioVue ™ AHG cards.Cell modified with the peptide-lipid construct M17 provided the most“false positive” reactions with negative serums. The reactivity of the 6most false positive samples when tested against other modified cellsshows that some are unreactive (M28, M27), some are poorly reactive orshow a single discrete reactivity (M5, M29, M19) while others are morereactive (M31, M26). Minor changes in amino acid sequence can influencethe rate of false positive reactivity. Cells modified with theconstructs M30 and M28 show both specificity and low non-specificity. Tseries reactives (n = 58) % 2 4 31 44 61 18 21 28 55 42 63 62 7 20 48 2239 23 30 positive M17 10 8 8 10 10 12 12 12 12 10 10 8 8 8 8 8 5 3 333%  M28 10 8 8 10 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 9% M30 10 8 8 10 10 00 0 0 0 0 0 0 0 0 0 0 0 0 9% 39 T negative samples were negative withall 3 constructs. Larger series = 102 T18 T21 T28 T55 T78 T92 30 Mur 9 DT Y P A H T A N E — — — — — — 17 Mur 2 T Y P A H T A N E V 12 12 12 1212 12 28 Mur 6 T Y P A H T A N E — — — — — — 27 Mur 5 T Y P A H T A N —— — — — — 25 Mur 4 Y P A H T A N E — — — —  5 — 26 Mur 7 Y P A H T A N EV  5 10 12 10 10 12 31 Mur 10 Y P A H T A N E V S — 10 12 —  8 12 19 Mur3 P A H T A N E V — — — 12 — — 29 Mur 8 P A H T A N E V S — —  5 — —  85 samples reacted with all 3 constructs Panel Serum #7 False (T8445)Positivity 30 Mur 9 D T Y P A H T A N E 8 − 17 Mur 2 T Y P A H T A N E V10  +++ 28 Mur 6 T Y P A H T A N E 8 − 27 Mur 5 T Y P A H T A N — − 25Mur 4 Y P A H T A N E 3 26 Mur 7 Y P A H T A N E V 10  +++ 31 Mur 10 Y PA H T A N E V S 8 ++ 19 Mur 3 P A H T A N E V 8 + 29 Mur 8 P A H T A N EV S 8 +

TABLE 20 Sera reactive with RBCs modified to incorporate the M1 peptide-lipid construct or M2 peptide-lipid construct constructs by contactingthe cells with a 500 μg/ml dispersion of the construct (Method 1) were“neutralised” with the peptide QTNDKHKRDTY and retested against themodified cells. Sera were neutralized by adding 10 μL of 1 mg/mlsolution of peptide to a 50 μL volume of sera and incubating for 30minutes at 37° C. Testing was performed using BioVue ™ cards. M1modified cells M2 cells vs serum Identity of sera #4 #5 #6 #2 #6 #8Serum alone 5 5 10 8 8 8 Serum + peptide 0 0 0 0 2 0

TABLE 21 Sera reactive with RBCs modified to incorporate the M13peptide- lipid construct by contacting the cells with a 500 μg/mldispersion of the construct (Method 1) were “neutralised” with thepeptide SSQTNDKHKRDTY and retested against the modified cells. Sera wereneutralized by adding 10 μL of 1 mg/ml solution of peptide to a 50 μLvolume of sera and incubating for 30 minutes at 37° C. Testing wasperformed using BioVue ™ cards. M13 modified cells Identity of sera #3#42 #37 #34 Serum alone 8 8 8 8 Serum + peptide 0 0 0 0

TABLE 22 Cells were modified by the peptide-lipid construct M22 (2hours, 37° C.) and two positive reactions were identified.Neutralisation experiments were then performed. A volume of 40 μl ofplasma was incubated with 10 μl of peptide or Ac—C at a concentration of1.0 mg/ml for 30 minutes at 37° C. The standard AHG test in BioVue ™ wasthen performed. The false positive reaction for PAC74 was confirmed as areaction not neutralised by addition of peptide. The true positivereaction for TAP1 confirmed as reaction neutralised by peptide and thewhole construct, but not the construct bearing only acetylated cysteine.PAC74 TAP1 Pre-neutralisation serology 8 8 (cells modified with thepeptide-lipid construct M22) Post-neutralisation serology (cellsmodified with the Neutraliser F Linker peptide-lipid construct M22) nilnil nil 8 8 M22 peptide SQTNDKHKRDC nil 8 — M28 peptide TYPAHTANEC nil 88 M22 molecule SQTNDKHKRDC CMG(2) — — Cys-CMG-DE Ac—C CMG(2) — 8VMYASSG? 8 8

TABLE 23 Cells were modified by the peptide-lipid construct M28 (2hours, 37° C.) and four positive reactions were identified.Neutralisation experiments were then performed. A volume of 40 μl ofplasma was incubated with 10 μl of peptide or Ac—C at a concentration of1.0 mg/ml for 30 minutes at 37° C. The standard AHG test in BioVue ™ wasthen performed. The neutralisations of PAC70, 71 and 72 with the M28peptide suggests specificity. The fact that the unrelated peptide M22was also able to cause neutralisation of serums PAC71 and PAC72,together with reductions in score with other unrelated structures,revises the results for these two sera as being false positivereactivity. The fact that PAC70 does not react with Miltenbergerpositive cells suggests that although an antibody appears to be presentto the peptide sequence it is not blood group specific. In contrastalthough TAP2 was not fully inhibited by peptide, the substantialreduction in score suggests specificity, although it is possible thatspecificity may be present with a low level of non-specificity assuggested by the reaction score reduction against Cys-CMG-DE. PAC70PAC71 PAC72 TAP2 Pre-neutralisation serology 8 5 5 8 (cells modifiedwith the peptide-lipid construct M28) Post-neutralisation serology(cells modified with the Neutraliser F group Linker peptide-lipidconstruct M22) saline nil nil 8 5 5 8 M22 peptide SQTNDKHKRDC nil 8 — —3 M28 peptide TYPAHTANEC nil — — — 8 Cys-CMG-DE Ac—C CMG(2) 8 3 3 5Atri-CMG-DE GalNAcα3[Fucα2]Galβ CMG(2) 8 3 5 8 Atri-adipate-DEGalNAcα3[Fucα2]Galβ adipate 8 3 5 8 VMYASSG? 8 5 5 8

Consideration of the MUT peptide reactivities presented in the foregoingin Tables shows that peptides M22, M36 and M37 all showed superiorsensitivity and specificity towards a human polyclonal antibody panelwhen compared with sequence 1, the sequence identified in the prior art(Reid and Lomas-Francis (2004)).

Modification of Red Blood Cells with Peptide-Lipid Constructs withPeptide in Alternative Configurations

Peptide-lipid constructs comprising CMG(2) and the following peptideswere prepared:

ThrTyrProAlaHisThrAlaAsnGluCys (M44) and CysThrTyrProAlaHisThrAlaAsnGlu(M45)

The termini of the peptides were formylated and amidated to provide“capped” peptides. The reactivity of random Miltenberger antibodypositive samples and three false positive antibody negative samples weretested against RBCs modified to incorporate these “capped” peptide-lipidconstructs (50 μg/mL, 2 hours at 37° C.).

The reactivity was assessed and recorded in Table 24. The capping of thepeptide did not affect reactivity with positive samples, nor did itappear to affect reactivity with false-positive antibody-negativesamples. However, linkage of the peptide via a Cys residue located atthe amino terminus (as opposed to the carboxy terminus) appeared toreduce the likelihood of reactivity with the false-positive antibodynegative samples and improve reactivity with the Known antibody positivesamples.

Whilst not wishing to be bound by theory it is speculated that thepresentation of the peptide by cells modified to incorporate M45 may bemore analogous to the presentation of the corresponding peptide sequenceexpressed by the naturally occurring antigen.

TABLE 24 Natural Not MiIII Sample M44 M45 modified cells Antibody 71 1010 0 8 positive 67 8 10 0 5 serums 65 10 8 0 12 55 8 8 0 10 61 8 5 0 8942-433813-3 0 5 0 12 68 0 0 0 10 False- PAC302 5 0 0 0 positive PAC33212 0 0 0 antibody PAC340 5 0 0 0 negative serumsModification of Cells and Multi-Cellular Structures with Biotin-LipidConstructs

The modification of red blood cells (RBCs) and murine embryos by theconstruct designated Biotin-CMG(2)-Ad-DOPE was demonstrated usingAvidin-Alexafluor (Avidin AF).

Materials

A stock solution of the construct designated Biotin-CMG(2)-Ad-DOPE (100μL) was prepared in water at a concentration of 10 mg/mL. A stocksolution of Avidin-Alexafluor (Avidin-AF) was prepared in sterilephosphate buttered saline (PBS) at a concentration of 2 mg/mL. A stocksolution of biotinylated gangliocide (BioG) was prepared in sterile PBSat a concentration of 5 mg/mL.

Red Blood Cells

Dilution series of the construct designated Biotin-CMG(2)-Ad-DOPE andBioG (positive control) were prepared at concentrations of 0.001, 0.1,0.1 and 1 mg/mL with Celpresol™. O group red blood cells (RBCs) weremodified by incubation of 15 μL of packed RBCs and 5 μL of a dilution ofthe construct designated biotin-CMG(2)-Ad-DOPE or BioG. Incubations wereperformed in a plastic ependorf tube of nominal volume 1.5 mL for 2hours at 37° C. in a water bath. O group RBCs were incubated with asolution of BioG at a concentration of 0.33 mg/mL as a positive control.

Incubated RBCs were washed 3 times with PBS in a mini centrifuge.Fluorescent labeling of the washed, modified RBCs was performed byadding 10 μL of a solution of Avidin-AF at a concentration of 0.1 mg/mL.The RBCs were then incubated in the dark for 1 hour at 37° C. in a waterbath and washed 3 times with PBS.

85% solution of the washed, fluorescent labeled RBCs were viewed on aslide with cover slip under a fluorescent microscope at 488 nm andphotographic exposure of 1.903 (FIG. 6). The intensity of thefluorescent signal was recorded using a scoring system of 0 (nofluorescence observed) to 4 (maximum fluorescence observed).

An aliquot of the washed, modified RBCs obtained from incubation withthe construct designated biotin-CMG(2)-Ad-DOPE at a concentration of 1mg/mL was retained and stored at 14° C. for 14 days. Retention of theconstruct by the RBCs was assessed by incubation with Avidin-AF asbefore.

The assessment of the fluorescence is recorded in Table 25.

TABLE 25 Dilution (mg/ml) 1 0.1 0.01 0.001 0 Biotin-CMG(2)-Ad-DOPE 4 2 10 0 Day 0 Biotin-CMG(2)-Ad-DOPE 4 — — — — Day 14 BioG 3 2 0 0 0 Day 0

Murine Embryos

Murine embryos (morula/early blastocyst stage, 3.5 day) were incubatedin 50 microlitre microdrops in Blastassist™ culture media. Embryos wereincubated with construct designated biotin-CMG(2)-Ad-DOPE at aconcentration of 0.1, 1 or 2 mg/mL or BioG at a concentration of 0.5mg/mL (positive control).

The zona pellucida of the embryos was removed by treatment with 0.5%pronase (4 minute incubation) and washing 3 times in embryo handlingmedia prior to introduction into the microdrops. Each microdrop wasequilibrated 5% CO₂ at 37° C. overnight prior to introduction of theembryos.

Microdrops containing embryos and the construct designatedbiotin-CMG(2)-Ad-DOPE were incubated for 2 hours in 5% CO₂ at 37° C.Microdrops containing embryos and BioG were incubated for 40 minutes in5% CO₂ at 37° C.

Following incubation each group of embryos was washed 3 times inhandling media and transferred to 50 microlitre microdrops containing 2mg/mL Avidin-AF microdrops for fluorescent labelling. The microdropscontaining transferred embryos were incubated at 37° C. for 30 minutesin the dark.

Each group of embryos was then washed 3 times in handling media andmounted on a glass microscope slide for viewing. Embryos were viewedunder a fluorescent microscope at 488 nm. The intensity of thefluorescence was recorded using a scoring system of 0 (no fluorescence)to 4 (maximum fluorescence).

TABLE 26 Biotin- Biotin- Biotin- CMG(2)-Ad- CMG(2)-Ad- CMG(2)-Ad- DOPEDOPE DOPE BioG Media 0.1 mg/mL 1 mg/mL 2 mg/mL 0.5 mg/mL alone 2 1 1 3 1n = 21 n = 19 n = 19 n = 19 n = 19

Immobilization of Spermatozoa and Cells

The immobilization of spermatozoa and red blood cells (RBCs) wasdemonstrated by use of the construct designated Biotin-CMG(2)-Ad-DOPEand streptavidin beads (Dynabeads® M-280).

Materials

A stock solution of the construct designated Biotin-CMG(2)-Ad-DOPE (100μL) was prepared in water at a concentration of 10 mg/mL and diluted inculture media (Medicult 10310060A) to provide a test dilution at 0.1mg/mL.

The spermatozoa in fresh semen (less than one day old) were assessed formotility (80%, grade 3 (fast, forward progression) by 10-fold dilutionin culture medium (Medicult 10310060A; pre-incubated for a minimum of 2hours at 37° C. in a 5% CO₂ atmosphere). Spermatozoa counts(91.5×10⁶/mL) were performed by 10-fold dilution in deionised water.

Spermatozoa were washed and isolated by layering 1.1 mL of fresh semenover a gradient of SpermGrad 125 (Vitrolife 10099; 2 mL of 40% solutionover 2 mL of 80% solution in a 15 mL round bottom tube) and centrifugingat 500×g for 20 min.

The bottom layer of the gradient (c. 0.7 mL was transferred to 4 mLround bottom tubes and c. 2 mL flushing (handling) media (Medicult10840125A) added. The tube was centrifuged at 300×g for 10 min and thespermatozoa washed two more times (mixing by tube inversion).

Samples of washed spermatozoa were incubated overnight at 37° C. in a 5%CO₂ atmosphere. Spermatozoa counts (c. 25×10⁶/mL) were performed postovernight incubation by 10-fold dilution in deionised water.

Spermatozoa

A volume of 100 μl of the test dilution of the construct designatedBiotin-CMG(2)-Ad-DOPE (I) was added to each of four 0.6 mL ependorftubes (A-D) and 100 μL of culture media added to one 0.6 mL ependorftube (E).

Open tubes were incubated at 37° C. in a 5% CO₂ atmosphere prior toaddition of c. 70 μL spermatozoa (c. 25×10⁶/mL) to each tube andincubation for 120 min (A), 60 min (B), 30 min (C), 10 min (B) and 120min (E).

Following incubation a couple of drops of flushing media were added andthe tubes centrifuged at 300×g for 5 min. The spermatozoa were washedtwo more times with flushing media and before being re-suspended inculture media to a final volume of 100 μL.

Streptavidin beads at a concentration of c. 6.25×10⁶/100 μL were diluted35 times in BSA plus flushing media to provide a ratio of 0.1beads/spermatozoa when mixed in equal volume with a diluted suspensionof the modified spermatozoa.

A volume of 5 μL of a diluted suspension of the modified spermatozoa wasmixed on a slide with 5 μL of a diluted suspension of streptavidin beadsand covered with a coverslip.

The mixture was observed under a microscope at 400× magnification. FIG.9 provide a photomicrograph of the mixture provided following incubationwith 0.1 mg/mL of the construct designated Biotin-CMG(2)-Ad-DOPE for 60min (B).

The assessment of the attachment of streptavidin beads to modifiedspermatozoa is recorded in Table 27.

TABLE 27 Biotin-CMG(2)- Number of beads attached Ad-DOPE (I) Incubationto spermatozoa treatment time (min) Immediate 30 min A 120 10-12 10-15(with cross-linking) B 460 2, 4, 8 8 C 30 1-3 3 D 10 1 1 E (Control) 1200 0

Spermatozoa were observed to retain motile capacity despite attachmentof beads (no acrosome reaction was evident) with a preference ofattachment to motile spermatozoa.

Red Blood Cells

A dilution of the construct designated Biotin-CMG(2)-Ad-DOPE wasprepared at a concentration of 1 mg/mL with Celpresol™. A volume of 60μL washed A group red blood cells (RBCs) was modified by incubation with20 μL of the dilution of the construct at 37° C. for 2 hours.

The modified RBCs were washed twice in PBS and once in Celpresol™ asdescribed above. A 2% cell suspension of washed cells (modified orcontrol) was prepared in Celpresol™ and cell concentration (150×10⁶/mL)determined using a haemocytometer. Similarly the concentration of asuspension streptavidin beads (134×10⁶/mL) was determined.

A volume of 50 μL of the suspension of streptavidin beads was added tothe wells of a 96-well plat with a Neodymium (Rare-Earth) Super magnet(Magnets NZ Limited) affixed to the base. A volume of 50 μL of asuspension of RBCs was added to provide a bead to RBC ratio of c. 1:1and incubated at room temperature for 1 hour to allow RBCs to settle.

The wells were washed 3× with PBS, aspirating the washing solution witha pipette. The washed wells were observed under a microscope and theRBCs determined to be retained (FIG. 10).

Separation of Populations of Cells

A 0.5 mg/ml solution of the construct designated Biotin-CMG(2)-Ad-DOPEwas prepared in Celpresol™ and a volume of 10 μl used to modify 30 μlpacked cell volume of group 0 RBCs in a 1 ml eppendorf tube to provide afirst population of cells. Unmodified group A RBCs were used as a secondpopulation of cells.

Both populations of cells were incubated for 37° C. for 2 hrs in a waterbath and then washed 2× in PBS and 1× in Celpresol using an Immufuge II(low, 1 min). The concentration of cells in each suspension was made upto 2% by adding 1.5 mL.

The suspensions of RBCs were mixed with avidinylated magnetic Dynabeadsat an approximate ratio of RBC:bead of 1 and incubated for 10 min atroom temperature on a gyrator. Samples of the first and secondpopulations of RBCs were then mixed in equal volumes (35 μl each) in anependorf tube for two minutes.

The contents of the ependort were transferred to the well of a 96-wellplate and a magnet was applied to the underside of the well for 1minute. The supernatant was carefully removed with the magnet appliedand without disruption of the beads. The blood grouping of the cells ofthe supernatants were then assessed by applying 30 μl of supernatant and30 μl anti-A antibody to a Dynamed™ gel card. Cards were spun for 10 minin a centrifuge. Retention of the O group RBCs by the magnet wasdemonstrated by the absence of a pellet of group O cells.

Modification of Cell Layers with Biotin-Lipid Constructs

The modification of monolayers of the cell line RL95-2 (established froma human endometrial adenocarcinoma (ATCC HTB CRL 1671)) in serum-freeand serum-containing media was evaluated.

D-MEM/F12 (Gibco 11320-033, Invitrogen NZ) containing 1%penicillin/streptomycin (Gibco 15140-122, Invitrogen NZ) was used as aserum-free medium. D-MEM/F12 10% FBS (Gibco 10091-130, Invitrogen NZ)containing 1% penicillin/streptomycin and 5 μg/mL insulin (Gibco12585-014, Invitrogen NZ) was used as a serum-containing medium.

A suspension of the cell line RL95-2 was diluted in pre-warmedserum-containing media to the required concentration e.g. 4×10⁵cells/mL. A 25 μL volume of the suspension was used to seed the requiredwells in a Terasaki tray so that each treatment was performed induplicate. The plates were incubated overnight in a 5% CO₂, 37° C.incubator until the monolayer was approximately 60% confluent.

Dilutions of Biotin-CMG(2)-Ad-DOPE were prepared and 12 μl volumes addedto wells containing washed cell layers to provide final concentrationsof 20, 100 or 500 μg/mL. Trays were incubated at 37° C., 5% CO₂ for 120min.

The cells were then washed and a 12 μl volume of a 0.1 mg/mL solution ofAvidin Alexa Fluor® 488 added. The cells were then incubated at roomtemperature for a further 30 minutes in the dark.

The monolayers were finally washed 3 times with PBS, the trays invertedand photographed using an Olympus BX51 fluorescent microscope at 200×magnification, exposure time 475 ms (FIG. 12).

When the construct is inserted in serum-free media at 20, 100 and 500μg/mL, a homogenous intense fluorescent signal is observed in the cellmembrane that intensifies with increasing concentration of the construct(FIGS. 12A, C and E). When the construct is inserted in serum-containingmedia, weakened fluorescence is observed at the same concentrations(FIGS. 12B, D and F). These results imply that optimal insertion ofconstruct into the cell membranes requires serum-free media.

When the construct is inserted in serum-free media at 20, 100 and 500μg/mL, an homogenous intense fluorescent signal is observed in the cellmembrane that intensifies with increasing concentration of construct.The intensity of the fluorescence also increased with increasinginsertion time (Table 28).

These results imply that optimal insertion of construct into cellmembranes occurs with increased concentration of construct and/orincreased insertion time.

TABLE 28 Optimal insertion of construct into cell membranes occurs withincreased concentration of construct and/or increased insertion time.Mean Fluorescence* Concentration of biotin- Insertion timeCMG(2)-Ad-DOPE (μg/mL) 10 30 60 120 20 1+ 1-2+ 2-3+ 3-4+ 100 2+ 3 3+ 4+500 3+ 3-4+ na na Media alone 0  0 0  0  ‘na’ denotes “not assessed”.

When avidin Alexa Fluor® 488 was added to construct modified RL95-2cells and incubated at 37° C. for 4 and 24 hr the fluorescence graduallyshifted from the cell surface to the cell interior (FIG. 13). Nointernalization was observed when cells were incubated at 4° C.

Fluorescence was detected in construct modified RL95-2 cells 24 hrpost-insertion when cultured in serum-free media, albeit with reducedfluorescence from T=0 (Table 29). However, when cells were cultured inserum-containing media a fluorescence score of 1+ was detected at thehighest concentration of construct (500 mg/mL), but not at lower. Theseresults imply that the construct is optimally retained in cell membranes24 hr post-insertion when cultured in serum-free media, but not inserum-containing media.

TABLE 29 Retention of construct 24 hours post-insertion. The constructwas detected in cells cultured for 24 hr post-insertion in serum-freemedia, but was only detected at the highest concentration inserum-containing media. Concentration of biotin- Mean Fluorescence*CMG(2)-Ad-DOPE T = 24 hr (μg/mL) T = 0 Serum-free Serum-containing 203-4+ 1-2+ 0 100 4+ 2-3+ 0 500 4+  1+ Media alone 0  0  0 ‘na’ denotes“not assessed”.

Modification of Antigen Presentation

The amount of construct used in the manufacture of quality controlscells as described in the specification accompanying internationalapplication no. PCT/NZ2005/000052 (publication no. WO 2005/090368) is adeterminant of the cost of manufacture.

It was anticipated that presentation of antigen at a distance from theimmediate milieu of the cell surface may promote recognition bycross-reactive antibody and subsequent agglutination. The estimateddistances from the cell surface for an antigen (F) of a functional lipidconstruct (F-S-L) where the spacer (S) includes the structural motif ofthe present invention are 7.2 nm (CMG(2)) and 11.5 nm (CMG(4)). Thesedistances compare with 1.9 nm for the antigen of a construct (F-S-L)where the spacer is one described in the specification accompanyinginternational application no. PCT/NZ2005/000052 (publication no. WO2005/090368), i.e. A_(tri)-sp-Ad-DOPE (1).

To test the hypothesis solutions of four different trisaccharide(A_(tri))-lipid constructs were prepared as 50 μM, 10 μM and 5 μMsolutions in Celpresol™. Modified red blood cells were then prepared bycontacting 0.6 mls (pcv) of washed RBCs with 0.6 mls of the relevantsolution. The mixtures were incubated at 37° C. for 2 hours and thenwashed 3 times to provide a modified cell suspension.

The modified cell suspensions were prepared in 0.8% in Celpresol LISS™suitable for BioVue serology cards. A volume of 0.05 mls of CSLmonoclonal anti-A (026129801) followed by 0.05 mls of modified cellsuspension was applied to each card. Reactions were recorded followingcentrifugation in a BioVue cassette centrifuge (Table 29).

A comparison of the recorded reaction for molar equivalents of theA_(tri)-lipid constructs demonstrates that less CMG(2) or CMG(4)construct is required to provide an equivalent serological result. Therewas no significant gain with CMG(2) compared with CMG(4). There was aminor, but not significant change for MCMG (2).

Although the invention has been described by way of examples it shouldbe appreciated that variations and modifications may be made to theclaimed methods without departing from the scope of the invention. Asnoted it will be understood that for a non-specific interaction, such asthe interaction between the diacyl- or dialkyl-glycerolipid portion ofthe functional-lipid constructs and a membrane, structural andstereo-isomers of naturally occurring lipids can be functionallyequivalent.

Where known equivalents exist to specific features, such equivalents areincorporated as if specifically referred to in this specification. Forexample, it is contemplated that diacylglycerol 2-phosphate could besubstituted for phosphatidate (diacylglycerol 3-phosphate) and that theabsolute configuration of phosphatidate could be either R or S.

TABLE 30 A_(tri)-lipid construct Structural motif Conc Serologicalresult at reciprocal of dilution of anti-A of spacer (μM) Neat 2 4 8 1632 64 128 256 512 1024 0 Adipate 50 12 12 12 12 12 12 10 8 8 3 — — 10 1010 10 8 8 8 8 3 — — — — 5 8 8 5 3 — — — — — — — — CMG(2) 50 12 12 12 1212 12 10 10 8 5 3 — 10 12 12 12 10 10 10 10 8 5 3 — — 5 10 10 10 10 8 88 5 3 — — — mCMG(2) 50 12 12 12 12 12 12 12 10 10 8 5 — 10 12 12 12 1212 10 10 8 8 5 — — 5 10 10 10 10 10 8 8 5 3 — — — CMG(4) 50 12 12 12 1212 12 10 10 8 5 3 — 10 12 12 12 10 10 10 10 8 8 3 — — 5 10 10 10 10 8 88 5 3 — — —

REFERENCES

-   Audibert et al (1987) Conjugates of haptenes and muramyl-peptides,    endowed with immunogenic activity and compositions containing them    U.S. Pat. No. 4,639,512.-   Blume et al (1993) Specific targeting with poly(thylene    glycol)-modified liposomes coupling of homing devices to the ends of    the polymeric chains combines effective target binding with long    circulation times. Biochimica et Biophysica Acto, 1149: 180-184-   Bovin et al (1993) Synthesis of polymeric neoglycoconjugates based    on N-substituted polyacrylamides Glycoconjugate Journal 10, 142-151.-   Bovin et al (2005) Synthetic membrane anchors International    application no. PCT/NZ2005/000052 (publ. no. WO 2005/090368).-   Carter et al (2007) Cell Surface Coating with Hyaluronic Acid    Oligomer Derivative International application no. PCT/NZ2006/000245    [publ. no. WO 2007/035116].-   Chung et al (2004) Casual Cell Surface Remodelling Using    Biocompatible Lipid-poly(ethylene glycol) (n): Development of    Stealth Cells and Monitoring of Cell Membrane Behaviour in    Serum-supplemented Conditions. J Biomed. Mater. Res, Part A,    70A/2:179-185-   Frame et al (2007) Synthetic glycolipid modification of red blood    cell membranes Transfusion, 47, 876-882.-   Galanina et al (1997) Further refinement of the description of the    ligand-binding characteristics for the galactoside-binding mistletoe    lectin, a plant agglutin with immunomodulatory potency Journal of    Molecular Recognition, 10, 139-147.-   Haselgrübler et al (1995) Synthesis and Applications of a New    Poly(ethylene glycol) Derivative for the Crosslinking of Amines with    Thiols. Bioconjugate Chem, 6: 242-248-   Hashimoto et al (1986) Iodacetylated and biotinylated liposomes:    Effect of spacer length on sulfhydryl ligand binding and avidin    precipitability. Biochim Biophys Acta, 856: 556-565.-   Holmberg et al (2005) The Biotin-streptavidin interaction can be    reversibly broken using water at elevated temperatures,    Electrophoresis, 26 (3), 501 to 510.-   Ishida et al (2001) Liposomes Bearing Polytheneglycol-Coupled    Transferrin with Intracellular Targeting Property to the Solid    Tumors In Vivo. Pharmaceutical Research, 18 (7): 1042-1048-   Kato et al (2004) Rapid Proprotein anchoring into the membranes of    mammalian cells using olial chain and polyethylene glycol    derivatives.-   Kinsky et al (1983) An alternative procedure for the preparation of    immunogenic liposomal model membranes. J Immunol Method, 65: 295-306-   Korchagina and Bovin (1992) Synthesis of spacered trisaccharides    with blood group A and B specificities and fragments and structural    analogues of them Soviet Journal of Bioorganic Chemistry, Vol. 18,    No. 2, 153-165.-   Krylov et al (2007) Stereoselective synthesis of the 3-aminopropyl    glycosides of α-D-Xyl-(1→3)-β-D-Glc and    α-D-Xyl-(1→3)-α-D-Xyl-(1→3)-β-D-Glc and of their corresponding    N-octanoyl derivatives Synthesis, 20, 3147-3154.-   Kung and Redemann (1986) Synthesis of carboxyacyl derivatives of    phosphatidylethanolamine and use as an efficient method for    conjugation of protein to liposomes. Biochim Biophys Acta, 862:    435-439-   Lee and Lee (1997) Facile Synthesis of a High-Affinity Ligand for    Mammalian Hepatic Lectin Containing Three Terminal    N-Acetylgalactosamine Residues Bioconjugate Chem., 8, 762-765.-   Legler et al (2004) Differential insertion of GPI-anchored GFPs into    lipid rafts of live cells The FASEB Journal, Online article    10.1096/fj.03-1338fje-   Litherland and Mann (1938) The amino-derivatives of pentaerythritol    Part I. Preparation Journal of the Chemical Society, 1588-95.-   Mann and Litherland (1938) Nature, No. 3574, 789.-   Mannino et al (1993) Liposomes as adjuvants for peptides:    Preparation and use of immunogenic peptide-phospholipid complexes.    Liposome Technology: 167-184-   Martin and Papahadjopoulos (1982) Irreversible coupling of    immunoglobulin fragments to preformed vesicles. An improved method    for liposome targeting. J Biol Chem, 257: 286-288-   Martin et al (1990) Liposomes a Practical Approach, 163-182-   Massaguer et al (2001) Synthesis of RGD Containing Peptides.    Comparative Study of their Incorporation to the Surface of    5-Fluoruridine Loaded Liposomes. Journal of Liposome Research,    11(I):103-113-   McHugh et al (1995) Construction, purification, and functional    incorporation on tumor cells of glycoplipid-anchored human B7-1    (CD80) Proc. Natl. Acad. Sci. USA, 92: 8059-8063-   McNaught (1996) Nomenclature of carbohydrates Pure & App. Chem., 68,    No. 10, 1919-2008.-   Medof et al (1996) Cell-surface engineering with GPI-anchored    proteins The FASEB Journal, 10: 574-586-   Morandat et al (2002) Cholesterol-dependent insertion of    glycosylphosphatidylinositol-anchored enzyme Biochimica et    Biophysica Acta, 1564: 473-478-   New (1992) Liposomes: A Practical Approach-   Nifant'ev et al (1996) Selectin-receptors 4: synthesis of    tetrasaccharides sialyl Lewis A and sialyl Lewis X containing a    spacer group ^(1,2) J. Carbohydrate Chemistry, 15(8), 939-953.-   Paulsen et al (1978) Darstellung selektiv blockierter    2-azido-2-desoxy-_(d)-gluco-und-_(d)-galactophyranosylhalogenide:    Reaktivitat und ¹³ C-NMR-Spektren Carbohydrate Research, 64,    339-364.-   Pazynina et al (2002) Synthesis of histo blood-group antigens A and    B (type 2), xenoantigen Galα1-3Galβ1-4GlcNAc and related type 2    backbone oligosaccharides as haptens in spacered form Mendeleev    Commun. 12(4), 143-145.-   Pazynina et al (2003) Synthesis of complex 2-3    sialooligosaccharides, including sulfated and fucosylated ones,    using Neu5Acα2-3Gal as a building block Mendeleev Commun, 13(6),    245-248.-   Premkumar et al (2001) Properties of Exogenously Added GPI-Anchored    Proteins Following Their Incorporation Into Cells Journal of    Cellular Biochemistry, 82: 234-245-   Reid and Lomas-Francis (2004) The Blood Group Antigen facts book.    Elsevier Academic Press, Amsterdam, 2nd ed.-   Ronzon et al (2004) Insertion of    Glycosylphosphatidylinositol-Anchored Enzyme into Liposomes The    Journal of Membrane Biology, 197: 169-177-   Shek and Heath (1983) Immune response mediated by    liposome-associated protein antigens III Immunogenicity of bovine    serum albumin covelantly coupled to vesicle surface. Immunology, 50:    101-106-   Sherman et al (2001) Synthesis of Neu5Ac- and    Neu5Gc-α-(2→6′)-lactosamine 3-aminopropyl glycosides Carbohydrate    research 330, 445-458.-   Skountzou et al (2007) Incorporation of    Glycosylphosphatidylinositol-Anchored Granulocyte-Macrophage    Colony-Stimulating Factor or CD40 Ligand Enhances Immunogenecity of    Chimeric Simian Immunodeficiency Virus-Like Particles Journal of    Virology, 81, 3: 1083-1094-   Vodovozova et al (2000) Antitumour activity of cytotoxic liposomes    equipped with s electin ligand SiaLe _(x), in a mouse mammary    adenocarcinoma model European Journal of Cancer, 36, 942-949.-   Winger et al (1996) Lipopeptide conjugates: biomolecular building    blocks for receptor activating membrane-mimetic structures.    Biomaterials, 17: 437-441

1) A functional lipid construct of the structure:

where F is a glycan, h is the integer 1, 2, 3 or 4, x is the integer 2,3 or 4, y is the integer 1, 2 or 3, R₁ and R₂ are independently selectedfrom the group consisting of: alkyl or alkenyl substituents of the fattyacids trans-3-hexadecenoic acid, cis-5-hexadecenoic acid,cis-7-hexadecenoic acid, cis-9-hexadecenoic acid, cis-6-octadecenoicacid, cis-9-octadecenoic acid, trans-9-octadecenoic acid,trans-11-octadecenoic acid, cis-11-octadecenoic acid, cis-11-eicosenoicacid or cis-13-docsenoic acid and R₃ is O of a substituted hydroxyl ofthe glycan. 2) A construct of claim 1 where the glycan is selected fromthe group consisting of: GalNAcα1-3(Fucα1-2)Galβ-;Galα1-3(Fucα1-2)Galβ-; Xylα1-3Glcβ-; Xylα1-3Xylα1-3Glcβ-;Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ-; Galβ1-4(Fucα1-3)GlcNAcβ-;Fucα1-2Galβ1-4(Fucα1-3)GlcNAcβ-; Neu5Acα2-3Galβ1-3GalNAcα-;Neu5Acα2-3Galβ1-3(6-HSO₃)GalNAcα-; Neu5Acα2-3Galβ1-3GlcNAcβ-;Neu5Acα2-3Galβ1-3(6-HSO₃)GlcNAcβ-; Neu5Acα2-3Galβ-;GalNAcα1-3Galβ1-4GlcNAc-; Galα1-3Galβ1-4GlcNAc-; Galα1-4Galβ1-4Glc-;Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ-; GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ-;Galα1-3(Fucα1-2)Galα1-4GlcNAcβ-; Fucα1-2Galβ1-4GlcNAcβ-;Galα1-3Galβ1-4GlcNAcβ-; Galα1-4Galβ1-4GlcNAcβ-;Neu5Acα2-3Galβ1-4GlcNAcβ-; Neu5Acα2-3Galβ1-4(6-HSO₃)GlcNAcβ-;Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ;Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-;Neu5Ac-α-(2-6′)-Galβ1-4GlcNAcβ-; and Neu5Gc-α-(2-6′)-Galβ1-4GlcNAcβ-.